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Enhancement of noradrenergic neurotransmission in the nucleus of the solitary tract modulates memory storage processes
Brain Research 987 (2003) 164–175 www.elsevier.com / locate / brainres
Research report
Enhancement of noradrenergic neurotransmission in the nucleus of the solitary tract modulates memory storage processes Teiko Miyashita, Cedric L. Williams* Department of Psychology, University of Virginia, 102 Gilmer Hall, P.O. Box 400400, Charlottesville, VA 22904 -4400, USA Accepted 15 July 2003
Abstract These studies examined whether posttraining activation of a1-noradrenergic receptors in the nucleus tractus solitarius (NTS) influences neural processes that are involved in encoding information into memory. Different groups of male Sprague–Dawley rats were trained in two separate learning tasks. In experiment 1, rats were given either a control solution or the a1-noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, or 10 mg / 0.5 ml) directly into the NTS immediately after they were given a footshock (0.35 mA, 0.5 s) in the dark compartment of an inhibitory apparatus. In a retention test given 48 h later, groups that received either 5.0 or 10.0 mg of phenylephrine avoided the dark compartment for a significantly longer period of time than the PBS control group (P,0.05 and P,0.01, respectively). In experiment 2, identical doses of phenylephrine were infused in the NTS following footshock delivery in one alley of a Y-maze. Animals given either 1.0 or 5.0 mg of phenylephrine performed significantly better than PBS controls on several different measures that served as indices of retention. The results indicate that activation of a1-noradrenergic receptors in the NTS plays a critical role in the transmission of signals from the periphery to brain systems that process memory for emotionally significant experiences. 2003 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Memory; Arousal; NTS; a1-Noradrenergic receptor
1. Introduction The effectiveness in which significant or meaningful experiences are encoded into long-term memory by limbic brain structures is now known to be dependent in part, on input received from peripheral neural circuits. Peripheral nerve fibers play an important role in relaying information regarding the physiological state of an organism following exposure to emotionally arousing stimuli to brainstem structures that make direct or polysynaptic connections on limbic neurons. A cluster of neurons located in the caudal medulla known collectively as the nucleus tractus solitarius (NTS) has received a great deal of recent attention as a major contributor to this process during memory formation.
*Corresponding author. Fax: 11-434-982-4785. E-mail address: [email protected] (C.L. Williams). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03323-7
The anatomical relationship between NTS neurons that receive input from peripheral autonomic pathways and their projections to central limbic structures allows NTS neurons to play an important role in modulating memory processing for arousing events. NTS neurons receive information regarding heightened activity in the periphery from the afferent branch of the vagus nerve [15] which conveys information concerning changes in cardiovascular, respiratory, gastrointestinal, and neuroendocrine systems. In response to activation by vagal terminals, neurons in the NTS transmit neural signals to brain structures that become active during the initial stage of memory processing, such as the amygdala [34,26]. The capacity for vagal or NTS input to influence the amygdala has been demonstrated in studies showing that electrical stimulation of the vagus nerve elicits increased firing in the amygdala [25] and increases the number of amygdala neurons that stain for c-fos, a marker for neuronal activation [22]. Neuronal activity in amygdala is also elevated following electrical
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stimulation of the NTS, that is, the site of termination of vagal afferent fibers [27]. The involvement of peripheral neural circuitry and brainstem structures in influencing amygdala activity represents one mechanism by which physiological arousal regulates brain functions during memory formation. Experimental evidence from neurochemical studies using the in vivo microdialysis technique also indicate that NTS neurons play an important role in transmitting information from the periphery to limbic structures following behavioral states of emotional arousal. For example, a dose of the hormone epinephrine which improves memory [10] also produces a significant elevation in norepinephrine concentrations in the amygdala [33]. However, inactivation of the NTS with lidocaine blocks the increase in norepinephrine released in the amygdala following epinephrine administration [33]. In addition, direct activation of NTS cells with either glutamate [21] a neurochemical released from vagal afferents, or noradrenergic agonists such as epinephrine [5] or clenbuterol [32] have all been demonstrated to significantly increase norepinephrine release in the amygdala. Of equal importance is the finding that the doses of these compounds that affect norepinephrine output in the amygdala also significantly improve memory when given into the NTS following training in spatial, aversive, or discrimination learning tasks [21,5,32]. Taken together, these findings indicate that the improvement in memory processing that follows emotionally arousing events is mediated in part by the transmission of information regarding the physiological state of an organism to brain structures such as the amygdala. Although the contribution of NTS neurons in modulating memory has only recently been revealed, the neurochemical agents that mediate neurotransmission between vagal afferents that terminate in the NTS, and NTS neurons that project to the limbic structures has not been well elucidated. As of date it is known that upon peripheral activation elicited by physiological arousal, vagal terminals release glutamate onto NTS neurons [18]. Glutamate release in turn, has been shown to elevate norepinephrine levels in the NTS [29]. Similar interactions between glutamate and norepinephrine activity in the NTS have been reported from experiments that employed sympathomimetic drugs to elicit physiological arousal by elevating blood pressure. Systemic administration of these pharmacological agents not only increases glutamate release in the NTS, but also causes a significant increase in extracellular norepinephrine concentrations in this nucleus [6]. Findings from studies combining electrophysiology with double fluorescence immunocytochemistry have identified two classes of norepinephrine containing neurons in the caudal NTS. Neurons which stained for dopamine-b-hydroxylase, the enzyme involved in synthesizing norepinephrine, were classified as either large neurons, with axonal process that exited the NTS or smaller local circuit
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neurons that were postsynaptic to glutamate containing vagal terminals [16]. The local circuit neurons in turn, formed direct synapses with the larger A2 neurons found in the NTS. In this study, both types of noradrenergic neurons exhibited excitatory responses following electrical stimulation of afferent vagal fibers. Considered together, these individual findings suggest that the norepinephrinecontaining neurons of the NTS that have terminal processes innervating structures such as the amygdala may be activated by possibly two mechanisms. One may involve direct activation of these projection neurons by glutamate released from vagal afferents or they may be activated by norepinephrine secreted from local circuit neurons that are interspersed between vagal afferents and the A2 neurons. The likelihood that the first mechanism is involved in the memory storage process was recently confirmed with both behavioral and neurochemical findings [21]. However, there is less evidence indicating whether the norepinephrine elevation in the NTS observed following peripheral physiological arousal or subsequent to glutamate release from vagal afferents is an important component of the processes in which emotionally arousing experiences are encoded into memory storage. Thus, the present studies were conducted to address this shortcoming by examining whether noradrenergic activation of the NTS plays a functional role in affecting memory formation. In experiment 1, rats implanted with injection cannula bilaterally in the NTS were given footshock training in an inhibitory avoidance task. Following training, the rats were removed from the apparatus and given a bilateral injection into the NTS of either PBS or the selective a1-noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, or 10.0 mg / 0.5 ml). The consequences of activating NTS neurons which in turn project to and release norepinephrine in limbic structures were assessed 2 days later by measuring latencies to enter the compartment of the apparatus where footshock was administered during training. To assess whether the changes in memory that may be produced by activating the NTS are not limited to an emotionally aversive task, a separate group of animals were trained in an appetitively motivated Y-maze discrimination task. In this experiment, animals were trained to eat five pellets in the left alley and 10 pellets in the right alley of a Y-shaped maze. On the sixth day of training, after the animals consumed all pellets in both alleys and then animal returned to the right alley, a footshock was administered. Following footshock, PBS or phenylephrine (0.5, 1.0, 5.0, or 10.0 mg / 0.5 ml) was microinfused into the NTS. The animals’ capacity to discriminate between the arms of the maze where shock was or was not delivered was examined on retention tests given 24 and 48 h later. The results of demonstrate that a1-nordrenergic receptors in the NTS play an important role in facilitating synaptic transmission in neurons that project to and release norepinephrine in limbic structures that are involved in memory formation.
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2. Methods
2.1. Subjects One-hundred and ten male Sprague–Dawley rats (275– 300 g) obtained from Hilltop Laboratories (Scottesdale, PA, USA) were used in experiments 1 (n575) and 2 (n530). Upon arrival, the animals were individually housed in plastic cages and maintained on a standard 12-h light–dark cycle with lights on at 07:00 h. The animals were adapted to the vivarium for 7 days without being disturbed during which food and water were available ad libitum.
2.2. Surgery In preparation for surgery each rat was given atropine sulfate (0.2 mg / kg, i.p.; Vedco, St. Joseph, MO, USA) to aid in respiration and then anesthetized with sodium pentobarbital (50 mg / kg; Abbott, North Chicago, IL, USA). After anesthetized, a midline scalp incision was made, and 15-mm 25.0-gauge extra thin wall stainless steel guide cannulas (Small Parts, Miami Lakes, FL, USA) were implanted bilaterally 2 mm above the NTS (AP: 213.30; ML; 61.0 from bregma; DV: 25.6) according to the atlas of Paxinos and Watson [23]. The cannulas and two anchoring screws were affixed to the skull with dental cement, and the scalp was closed with autoclips. Stylets (15 mm, 00 insect dissection pins) were inserted into the injection cannulas to maintain cannula patency. Penicillin (0.1 ml, i.m.; Fort Dodge Pharmaceuticals, Fort Dodge, IA, USA) was administered immediately after surgery. The animals were maintained in a temperature-controlled chamber for approximately 1 h after surgery. For the following 7 days of recovery, the animals were not disturbed in the vivarium.
2.3. Training apparatus and procedures 2.3.1. Experiment 1: inhibitory avoidance training In experiment 1, the rats were trained in a trough-shaped two-compartment inhibitory avoidance apparatus (91 cm in length, 21 cm wide at the top and 6.4 cm at the bottom). A metal sliding door (14.5 cm in height) that opened by retracting into the floor separated a brightly illuminated (40 W) compartment made out of white opaque plexiglas (31 cm long) from a darker compartment constructed of stainless steel plates (60 cm long). On the day of training, the rats were transported to the laboratory from the vivarium approximately 1 h before training. During training, each rat was placed in the white compartment of the inhibitory avoidance apparatus facing the sliding door. After the rat turned 1808 away from this position, the
sliding door was lowered into the floor. Once the animal turned and faced the open entrance to the dark compartment, the timer was initiated, and the latency to enter the dark compartment with all four paws was recorded. Once the animal was fully inside the dark compartment, the sliding door was raised and a 0.45-mA footshock was administered through the metal plates for 0.5 s. The rat was then removed from the training apparatus and given an intra-NTS microinfusion of a phosphate-buffered solution (PBS; n515) or 0.5 (n511), 1.0 (n516), 5.0 (n517), or 10.0 mg (n516) of phenylephrine in 0.5 ml. The animals were returned to the vivarium 1 h after training. Forty-seven hours later the animals were returned to the experiment room and left undisturbed for 60 min prior to the retention test. During the retention test, the animal was placed in the illuminated compartment of the apparatus facing away from the sliding door. After turning 1808, the door was lowered, and the latency to enter the dark compartment with all four paws was recorded (maximum of 600 s) and used as an index of retention.
2.3.2. Experiment 2: Y-maze discrimination task In experiment 2, a trough-shaped Y-maze constructed of stainless steel was used to train animals in a discrimination task. The three alleys of the maze were each 49 cm long318.5 cm high. The floor and ceiling were 4 and 19 cm wide, respectively. The floor of each alley was constructed of two stainless steel plates that were separated lengthwise by a 0.5-cm gap. The floor plates and lower half portion of the inner walls of each alley were covered by three removable trough-shaped cardboard panels (Fig. 1A). The left and right alleys were illuminated by a light (40 W) positioned behind translucent white plexiglas at the end wall of each maze arm. Each alley also contained a plastic food well which protruded from the end wall 2 cm above the floor where food pellets were placed. The rats were placed on a weight maintenance schedule for 7–10 days prior to the beginning of training to reduce their body weight by 15%. This target weight was maintained for the duration of the experiment. The first 2 days of Y-maze training consisted of habituation in which all animals freely explored the maze for 5 min. During habituation and the next 5 days of training, the stainless steel floor of each maze alley was covered with the trough-shaped cardboard panels. After 2 days of habituation, the rats underwent one training trial per day for 6 consecutive days. On each trial, the rat was placed in the stem of the Y-maze facing the end wall and given up to 5 min to locate and consume 10 Noyes Formula A food pellets (45 mg each; P.J. Noyes, Lancaster, NH, USA) placed in the food well protruding from the rear wall of the right alley and five pellets placed in the food well in the left alley. A trial ended when all 15 food pellets were consumed. The subject was then left in the maze to explore
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the alleys for an additional 30 s before being returned to its home cage. Digital timers were used to record: (1) the initial latency to enter either alley at the start of training; (2) the number of seconds required to consume all pellets in the right and left alleys after entering; (3) the number of seconds the rats remained in an alley after pellets were consumed; (4) the cumulative time to consume all pellets, from the beginning of a trial; and (5) the time spent in the non-baited stem (start alley) of the Y-maze. On day 6 of training, the cardboard insert covering the floor of the maze was removed from only the right alley to expose the stainless steel footshock plates. As in the previous days of training, each rat was allowed to consume all food pellets placed in both the left and right alleys. However, when the rat returned to the right alley and approached the empty food well, a 0.35-mA footshock was administered for 0.5 s through the stainless steel plates by a shock generator (Model 82406, Lafayette Instruments, Lafayette, IN, USA). Immediately after the footshock, the rat was removed from the maze and randomly assigned to groups that received a bilateral intra-NTS infusion of either PBS (n58) or 0.5 (n56), 1.0 (n56), 5.0 (n56), 10.0 mg (n54) of phenylephrine in 0.5 ml. Memory for footshock delivery in the right alley was assessed on days 7 and 8 under two separate conditions. On the first retention test (day 7), the cardboard insert was returned to the right alley as during the first 5 days of training (Fig. 1A). Thus, the stainless steel plates from which footshock was delivered on day 6 were concealed from the subjects on this retention test. This manipulation provided an assessment of the subjects’ memory of the footshock training in the absence of the most salient contextual cue associated with the footshock (i.e. the footshock plates). For the second retention test, given on day 8, the cardboard panel in the right alley was again removed to expose the stainless steel plates. On this day, the rats were exposed to the same contextual cues that were present on the day of footshock delivery (Fig. 1B). The two different retention tests were expected to reveal whether NTS activation with phenylephrine affects retention under conditions in which memory is weakened by removing salient cues that are linked or associated with an arousing events such as footshock. The tests were also designed to assess whether memory for an emotional experience is facilitated by activation of postsynaptic a1noradrenergic receptors in the NTS under normal testing conditions. The latency to first enter either alley and the number of seconds to consume all the pellets in the right alley served as indices of retention on both tests.
2.4. Microinjection procedures Immediately after training with footshock in either the inhibitory avoidance or Y-maze discrimination task, each animal was removed from the apparatus and gently restrained by hand in the experimenter’s lap. The stylets were removed and 17-mm 30-gauge injection needles were inserted through the injection cannulas into the NTS. The tips of the injection needles extended 2 mm beyond the base of cannulas at the NTS. The needles were connected to 10 ml Hamilton syringes via PE-20 polyethylene tubing. An automated syringe pump (Sage-Orion, Boston, MA, USA) delivered 0.5 ml of PBS (0.105 g of PO 4 NaH 2 H 2 O and 0.029 g of PO 4 Na 2 H in 100 ml of 9% saline) or 0.5, 1.0, 5.0, or 10.0 mg of phenylephrine (Sigma, St. Louis, MO, USA) into the NTS over a period of 60 s. After 0.5 ml of each test compound was delivered, the needles were retained in the cannulas for an additional 60 s to assure delivery of drugs. The stylets were then reinserted into the cannulas, and the animals were returned to their home cages.
2.5. Histology To verify correct placement of injection needle tips and guide cannulas, each animal was anesthetized with a euthanasia solution and perfused intracardially with saline (0.9%) followed by a 10% formalin solution. The brains were stored in formalin (10%) for approximately 7 days, sectioned on a freezing microtome in 40-mm sections, mounted on glass slides, and stained with cresyl violet. The location of the cannulas and injection needle tips were verified by examining enlarged projections of the slides. A photomicrograph of the injection needle tips in the NTS is displayed in Fig. 2.
2.6. Statistical analysis The behavioral measures from the inhibitory avoidance and Y-maze discrimination tasks are expressed as mean latency6standard errors (S.E.). Between-group comparisons for data collected during the inhibitory avoidance retention test were made with a one-way analysis of variance (ANOVA) followed by Fisher’s post-hoc tests. For the Y-maze discrimination task, each measure that served as indices of memory on day 7 and 8 was compared across the groups with ANOVA followed by Fisher’s post-hoc test. Repeated-measure ANOVA was also utilized to compare latencies across the training days (day 6–8) for
Fig. 1. Photographs of the Y-maze discrimination task used in experiment 2. (A) During habituation and days 1–5 of training, all alleys of the maze were covered with cardboard inserts as shown. On each day of training, five pellets were placed in the food cup at the end of the left maze alley and 10 pellets in the right alley. On day 6, the insert in the right alley was removed exposing the metal plate (B). After all 15 pellets were consumed and the animals returned to the right alley, a footshock (0.35 mA) was delivered for 0.5 s. On the retention test given 24 h after footshock training (day 7), the right alley was covered again concealing the cue that was most closely associated with footshock (A). During the second retention test (day 8), the insert was removed again to expose the metal plate from which footshock was delivered on day 6 (B).
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Fig. 2. A Photomicrograph illustrating placement of injection cannulas aimed above the NTS and the site of injection needle tips in the nucleus of the solitary tract (NTS). The shaded area represents the range of needle tip placements in the NTS.
each measure. Values of P,0.05 were considered significant in all tests.
3. Results
3.1. Experiment 1 Experiment 1 examined whether posttraining activation of a1-noradrenergic receptors in the NTS affects later retention performance. During the day of training and the retention test, the latencies to fully enter the dark compartment with all four paws were measured. A one-way ANOVA on the mean latencies to enter the dark compartment during training revealed no significant differences across the separate dose groups, F(4,70)50.84, P50.51. In the retention test however, there was a statistically significant difference in the mean latencies to enter the dark compartment, F(4,70)53.36, P,0.05. Post-hoc Fisher’s tests revealed that animals in the 5.0 and 10.0 mg phenylephrine groups spent a significantly longer period of time in the illuminated white compartment (M5143 and 166 s, respectively) before entering the dark compartment relative to the PBS controls (M559 s; P,0.05 and P, 0.01; see Fig. 3).
3.2. Experiment 2 The findings from experiment 1 indicate that infusion of the a1-noradrenergic agonist phenylephrine into the NTS
dose-dependently improves memory for footshock training. experiment 2 examined whether phenylephrine’s actions in the NTS are also manifested in a different type of learning condition requiring the animals to make a discrimination response. In experiment 2, animals were trained in an appetitively motivated Y-maze discrimination task and administered a control solution or one dose of the a1noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, 10.0 mg) into the NTS. On the first day of retention testing (day 7), the alley where footshock was delivered 24 h earlier (i.e. right alley) was covered with a cardboard insert. Even though this manipulation concealed the most obvious visual cue that was directly associated with the footshock, the performance of animals given a posttraining infusion of the a1noradrenergic agonist into the NTS was significantly different from that of controls on several measures. For example, a one-way ANOVA revealed that the latencies to first enter the right or left alley were significantly different among the separate dose groups, F(4,25)52.94, P,0.05. Post-hoc Fisher’s test indicated that 1.0 mg phenylephrine group took significantly longer to make an alley entry choice than the PBS control group (P,0.01; Fig. 4). Once the animal entered either alley, the length of time required to consume all food pellets at the end wall was measured. The separate drug groups did not differ in the time required to consume all food pellets in the alley where no footshock was given (i.e. left alley), F(4,25)5 1.27, P.0.05. This finding demonstrates that footshock administered in the right alley did not produce a global
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Fig. 3. Experiment 1. Mean (6S.E.) latencies to enter the dark compartment on the day of training (left graph) and during the retention test in an inhibitory avoidance learning task (right graph). All subjects had entrance latencies of less than 5 s on the training day. In contrast, the latencies to enter the dark compartment on the retention test given 48 h later was significantly longer in the groups given a posttraining injection of either 5.0 (n517) or 10.0 (n516) mg / 0.5 ml of phenylephrine in the NTS relative to controls that received PBS (phosphate buffered saline) (n515). * P,0.05, ** P,0.01.
aversion to the entire Y-maze, but only to the alley where this stimulus was delivered. In contrast, there was a significant overall difference in the time taken to consume food pellets in the right alley between the groups, F(4,25)52.85, P,0.05 (data not shown). For this measure, animals given 1.0 and 5.0 mg of phenylephrine into the NTS took significantly longer to eat food pellets than the PBS controls (P,0.05). The cumulative time to consume
all food pellets in the right alley from the start of the retention test was significantly longer in the 1.0 and 5.0 mg phenylephrine groups relative to controls (P,0.05; Fig. 5). Because some animals took a longer time to eat food in the right alley, the length of time they spend in other places of the Y-maze was also measured. Individual comparisons among the drug groups in the total length of time the animals spent in the left ‘safe’ alley during the retention
Fig. 4. Experiment 2. Mean (6S.E.) latencies to enter either the right or left alley of the Y-maze on retention test 1. The group given a posttraining injection of 1.0 mg / 0.5 ml of phenylephrine into the NTS on day 6 took significantly longer to make an entry choice after being placed in the stem of the Y-maze than the PBS control group. * P,0.01.
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Fig. 5. Experiment 2. Mean latencies (6S.E.) to consume all pellets in the right alley on day 6 (footshock training), day 7 (retention test 1 with footshock plates concealed), and day 8 (retention test 2 with footshock plates revealed). This measure represents the cumulative latency to consume all food pellets in the right alley of the Y-maze from the beginning of the trial. On the day of training, all groups consumed the 10 pellets the right alley in less than 31 s. On the retention test given 24 h following footshock and phenylephrine infusion into the NTS, subjects in the 1.0 and 5.0 mg phenylephrine group took significantly longer than the PBS controls to consume all 10 pellets in the right alley. Similarly, on day 8, subjects in the 1.0 mg phenylephrine group took significantly longer to consume the food pellets than those in the control group; * P,0.05.
test yielded significant between-group differences. As displayed in Fig. 6, animals in the 1.0 mg phenylephrine group remained in the left alley for a significantly longer
period of time than controls or the other dose groups (P,0.05). On the second day of retention testing, the animals were
Fig. 6. Experiment 2. Mean (6S.E.) length of time spent in the left ‘safe’ alley of the Y-maze on days 6–8 of training and retention testing. The length of time spent in the left alley while avoiding entry into the right alley was also measured. On the day of training, all animals spent less than 9 s in the left alley. During the first retention test conducted 24 h later, animals in the 1.0 mg / 0.5 ml phenylephrine group remained in the left alley significantly longer than the PBS group. * P,0.05. The difference in the amount of time spent in the left alley on the second day of retention was significant between PBS controls and the 1.0 mg and 5.0 mg phenylephrine groups * P,0.05.
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placed back in the Y-maze with the footshock plates of the right alley exposed. In contrast to the results from retention test 1, the latency to first enter either alley was not significantly different between the drug groups F(4,25)5 0.90, P5.47. However, there was a significant difference between PBS controls and the 1.0 mg phenylephrine group on the time taken to consume all food pellets in the right alley from the beginning of the retention test (P,0.05; Fig. 5). The same figure also displays the differences in this measure between the drug groups across the days of treatment and the two retention tests. As indicated, on the day of treatment, animals in all groups finished consuming food in the right alley at similar latencies (M513–30 s). Forty-eight hours following the footshock and drug treatment, the animals in the control group (M532.3 s) and lowest (0.5 mg; M542.7) and highest (10.0 mg; M562.5 s) phenylephrine dose groups consumed all pellets in the right alley within the same time frame as the day before. A repeated-measure ANOVA showed that the time taken to consume pellets in the right alley in the 1.0 and 5.0 mg groups was significantly longer on the retention test in comparison to the day of training (M5122.7, 98.3 s, respectively; P,0.05). A comparison of the length of time the animals remained in the left alley to avoid the exposed footshock plates in the right alley revealed that animals in the 1.0 and 5.0 mg phenylephrine groups remained there significantly longer during the retention test than those in the PBS group (P,0.05; Fig. 6). The length of time animals in the 1.0 mg phenylephrine groups remained in the non-baited stem of the Y-maze to avoid entry into the right alley was also significantly longer (P,0.05) than the
PBS controls (Fig. 7). The above findings indicate that activation of noradrenergic receptors in the NTS immediately following an emotionally arousing experience enhances later retention performance reflected by either increased avoidance response or decreased consummatory behavior such as feeding.
4. Discussion The findings from the present experiments demonstrate that posttraining activation of a1-noradrenergic receptors in the NTS improves memory under two separate types of learning conditions. In experiment 1, the a1-noradrenergic receptor agonist phenylephrine was administered bilaterally into the NTS following footshock administration in an inhibitory avoidance task. In the retention test given 48 h later, the latencies to enter the dark compartment for the 5.0 or 10.0 mg phenylephrine groups were significantly longer than those of the control group. This result suggests that activation of a1-noradrenergic postsynaptic receptors in the NTS with phenylephrine affects neural processes involved in consolidating memory for emotionally arousing experiences. The data from experiment 2 confirms and extends those from the first study by demonstrating that phenylephrine exerts similar effects on memory in a discrimination learning task. In the food-motivated Y-maze task, the performance of subjects given either 1.0 or 5.0 mg of phenylephrine was statistically different than that of the control groups on several measures used as indices of
Fig. 7. Experiment 2. Mean (6S.E.) length of time spent in the stem of the Y-maze (non-baited alley) on the second day of the retention test. Animals in the 1.0 mg phenylephrine group remained in the stem of the Y-maze significantly longer than the PBS (* P,0.05) controls while avoiding entry into the right alley.
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retention. Moreover, this pattern of responding was observed on both days of the retention test in spite of the changes made to remove contextual cues that were present only during the day of footshock delivery. These present findings expand the current understanding of specific classes of noradrenergic receptors in the NTS that play a critical role in facilitating information and encoding processes that culminates in better retention performance. Previously, simultaneous activation of both a- and bnoradrenergic receptors in the NTS was shown to influence memory storage processes following emotional arousal [5]. The results from the present studies provide evidence that sole activation of a1-noradrenergic receptors in the NTS is sufficient to modulate memory formation processes. The effects on memory produced by activating a1noradrenergic receptors in the NTS were apparent even in the first retention test in experiment 2 when the shock plates in the right alley were covered concealing the contextual cue that was directly related to the arousing footshock experience. Generally, contextual transformations of this kind where important cues that were present during learning are concealed during testing leads to poor retention performance in control subjects [30–32]. However, the present results seem to indicate that the changes in the learning environment did not influence the retention performance of animals administered 1.0 or 5.0 mg of phenylephrine. Animals in the 1.0 mg dose group took significantly longer to make a choice between entering the right or left alley upon being placed in the stem of the Y-maze than did those in the control group (Fig. 4). Even after the animals entered the covered right alley, those given 1.0 or 5.0 mg of phenylephrine took a significantly longer amount of time to approach and consume the food pellets located at the end wall. The prolonged avoidance of the right alley and food pellets can be interpreted as the animals’ exhibition of their memory for where and how the aversive experience took place and indicates that posttraining activation of a1-noradrenergic receptors in the NTS influences the strength in which new memory traces may be encoded. As a consequence, these new traces are less vulnerable to the influences of contextual changes that are distracting and detrimental to retention performance in non-treated animals. Another measure that served to reflect whether learning occurred following footshock delivery involved the shift in the animals’ alley preferences. The mean time spent in the left alley on the treatment day (day 6) was 6.3 s for PBS group and 7.7 s for 1.0 mg phenylephrine group. However, on the retention test (day 7) the mean latency increased to 26.0 s for the 1.0 mg group which was significantly longer than the mean length of time the controls remained (9.8 s) in this ‘safe’ area of the maze 24 h after footshock. If the circumstances surrounding footshock on the treatment day were significant enough to encode into memory, one would assume that upon exposure, the animals would adapt their behavioral response to avoid the footshock thereafter.
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On the second day of retention testing, the cardboard panel was removed in the right alley and the stainless steel footshock plates were exposed. As a result, the latencies to enter and consume food pellets in the right arm were longer than the previous day for all groups. Even though retention performance was more evident in all groups when the metal plates were visible, there were still significant differences between the control and 1.0 and 5.0 mg phenylephrine groups in the time taken to either consume all pellets in the right alley, or in the length of time the animals remained in the left alley or the stem of the Y-maze during the retention test. Thus, the animals in the 1.0 and 5.0 mg phenylephrine groups could discriminate between which alley is ‘safe’ relative to the alley of the maze where an unexpected and unpleasant event was experienced. Considered together with the findings from the first retention test, these patterns of contrasting behavior by the animals in the control and the 1.0 and 5.0 mg phenylephrine groups support a premise that increased noradrenergic neurotransmission in the NTS facilitates neural processes involved in encoding and storing information into memory. The results also indicate that activation of a1-noradrenergic receptors in the NTS is an important component in the mechanisms underlying modulation of emotional, contextual, and stimulus–response associations into memory storage processes. In experiment 2, the doses of phenylephrine that improved memory in the Y-maze discrimination learning task (1.0 and 5.0 mg) were lower than the effective doses in experiment 1 (5.0 and 10.0 mg). There are several possibilities for this finding. First, animals trained in the Y-maze task were on a food-restricted schedule for approximately 2 weeks prior to administration of the drugs into the NTS. Although the food deprivation procedure used to reduce weight by 15% does not alter the sensitivity to shock or acquisition of the avoidance response [20], it does elevate circulating concentrations of the hormone epinephrine in plasma [11]. Since a1-noradrenergic receptor activation in the NTS is one mechanism by which physiological signals from the periphery are transmitted to memory-encoding structures in the brain following emotional arousal, a lower dose of phenylephrine might be sufficient to produce an optimum memory enhancement with higher levels of epinephrine already circulating in the periphery. Another possible reason for a lower dose being effective in experiment 2 could have been the training procedure. In the inhibitory avoidance task, the footshock was administered in a novel training environment. In contrast, in the food-motivated discrimination task the animals were placed in the Y-maze for at least 60 s on each day of training for 5 days prior to the footshock. Since the aversive stimulus was never experienced during this period and only encountered on the day of treatment, it is possible that the animals associated the footshock with the context of right alley much more substantially with an element of surprise and elicited more arousal, and only a lower dose
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of phenylephrine was required to enhance memory retention in the task. The effects of phenylephrine infusion into the NTS on memory demonstrated in the present study are attributed to activation of a1-noradrenergic receptors localized on noradrenergic neurons in the NTS [8,15,17,13,14,28]. aand b-noradrenergic receptors in the NTS are likely to be activated by norepinephrine released from interneurons between vagal afferents and NTS neurons [16] or from noradrenergic neurons in the area postrema which also receives vagal input [2,4]. Therefore, an input to trigger norepinephrine release and receptor activation in the NTS could originate from signals carried by the vagus nerve arriving from the periphery regarding the physiological state of an organism. The afferent branches of the vagus nerve innervate a variety of visceral organs (for review, see Ref. [12]) and carry various types of signals including cardiovascular, respiratory, gastrointestinal, endocrine, autonomic, and immune to the brain via the NTS [1]. Therefore, information regarding changes in physiological state elicited by emotional arousal may be conveyed by the vagus nerve and generate alterations in the noradrenergic system in the NTS. In fact, an increase in blood pressure, one physiological response that may accompany emotional arousal, has been shown to elevate extracellular norepinephrine levels within the NTS [6]. Norepinephrine released in the NTS in response to peripheral activation stimulates NTS noradrenergic neurons that project to forebrain structures [26]. One of its termination sites is the amygdala [34,7,24], a limbic structure implicated in modulation of memory storage [3,19]. Norepinephrine release in the amygdala has been extensively established as a neural substrate for memory modulation elicited by peripheral arousal [9,33]. The results from the present study indicate that the A2 noradrenergic neurons in the NTS contribute to memory processing for emotionally arousing events. More specifically, catecholamines in the NTS and the activation of A2 neurons play an important role in insuring that the consequences of emotionally significant events are made more salient and encoded in the brain.
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Acknowledgements Research supported by the National Institute of Mental Health (MH 63343 to CLW).
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Research report
Enhancement of noradrenergic neurotransmission in the nucleus of the solitary tract modulates memory storage processes Teiko Miyashita, Cedric L. Williams* Department of Psychology, University of Virginia, 102 Gilmer Hall, P.O. Box 400400, Charlottesville, VA 22904 -4400, USA Accepted 15 July 2003
Abstract These studies examined whether posttraining activation of a1-noradrenergic receptors in the nucleus tractus solitarius (NTS) influences neural processes that are involved in encoding information into memory. Different groups of male Sprague–Dawley rats were trained in two separate learning tasks. In experiment 1, rats were given either a control solution or the a1-noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, or 10 mg / 0.5 ml) directly into the NTS immediately after they were given a footshock (0.35 mA, 0.5 s) in the dark compartment of an inhibitory apparatus. In a retention test given 48 h later, groups that received either 5.0 or 10.0 mg of phenylephrine avoided the dark compartment for a significantly longer period of time than the PBS control group (P,0.05 and P,0.01, respectively). In experiment 2, identical doses of phenylephrine were infused in the NTS following footshock delivery in one alley of a Y-maze. Animals given either 1.0 or 5.0 mg of phenylephrine performed significantly better than PBS controls on several different measures that served as indices of retention. The results indicate that activation of a1-noradrenergic receptors in the NTS plays a critical role in the transmission of signals from the periphery to brain systems that process memory for emotionally significant experiences. 2003 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Memory; Arousal; NTS; a1-Noradrenergic receptor
1. Introduction The effectiveness in which significant or meaningful experiences are encoded into long-term memory by limbic brain structures is now known to be dependent in part, on input received from peripheral neural circuits. Peripheral nerve fibers play an important role in relaying information regarding the physiological state of an organism following exposure to emotionally arousing stimuli to brainstem structures that make direct or polysynaptic connections on limbic neurons. A cluster of neurons located in the caudal medulla known collectively as the nucleus tractus solitarius (NTS) has received a great deal of recent attention as a major contributor to this process during memory formation.
*Corresponding author. Fax: 11-434-982-4785. E-mail address: [email protected] (C.L. Williams). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03323-7
The anatomical relationship between NTS neurons that receive input from peripheral autonomic pathways and their projections to central limbic structures allows NTS neurons to play an important role in modulating memory processing for arousing events. NTS neurons receive information regarding heightened activity in the periphery from the afferent branch of the vagus nerve [15] which conveys information concerning changes in cardiovascular, respiratory, gastrointestinal, and neuroendocrine systems. In response to activation by vagal terminals, neurons in the NTS transmit neural signals to brain structures that become active during the initial stage of memory processing, such as the amygdala [34,26]. The capacity for vagal or NTS input to influence the amygdala has been demonstrated in studies showing that electrical stimulation of the vagus nerve elicits increased firing in the amygdala [25] and increases the number of amygdala neurons that stain for c-fos, a marker for neuronal activation [22]. Neuronal activity in amygdala is also elevated following electrical
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stimulation of the NTS, that is, the site of termination of vagal afferent fibers [27]. The involvement of peripheral neural circuitry and brainstem structures in influencing amygdala activity represents one mechanism by which physiological arousal regulates brain functions during memory formation. Experimental evidence from neurochemical studies using the in vivo microdialysis technique also indicate that NTS neurons play an important role in transmitting information from the periphery to limbic structures following behavioral states of emotional arousal. For example, a dose of the hormone epinephrine which improves memory [10] also produces a significant elevation in norepinephrine concentrations in the amygdala [33]. However, inactivation of the NTS with lidocaine blocks the increase in norepinephrine released in the amygdala following epinephrine administration [33]. In addition, direct activation of NTS cells with either glutamate [21] a neurochemical released from vagal afferents, or noradrenergic agonists such as epinephrine [5] or clenbuterol [32] have all been demonstrated to significantly increase norepinephrine release in the amygdala. Of equal importance is the finding that the doses of these compounds that affect norepinephrine output in the amygdala also significantly improve memory when given into the NTS following training in spatial, aversive, or discrimination learning tasks [21,5,32]. Taken together, these findings indicate that the improvement in memory processing that follows emotionally arousing events is mediated in part by the transmission of information regarding the physiological state of an organism to brain structures such as the amygdala. Although the contribution of NTS neurons in modulating memory has only recently been revealed, the neurochemical agents that mediate neurotransmission between vagal afferents that terminate in the NTS, and NTS neurons that project to the limbic structures has not been well elucidated. As of date it is known that upon peripheral activation elicited by physiological arousal, vagal terminals release glutamate onto NTS neurons [18]. Glutamate release in turn, has been shown to elevate norepinephrine levels in the NTS [29]. Similar interactions between glutamate and norepinephrine activity in the NTS have been reported from experiments that employed sympathomimetic drugs to elicit physiological arousal by elevating blood pressure. Systemic administration of these pharmacological agents not only increases glutamate release in the NTS, but also causes a significant increase in extracellular norepinephrine concentrations in this nucleus [6]. Findings from studies combining electrophysiology with double fluorescence immunocytochemistry have identified two classes of norepinephrine containing neurons in the caudal NTS. Neurons which stained for dopamine-b-hydroxylase, the enzyme involved in synthesizing norepinephrine, were classified as either large neurons, with axonal process that exited the NTS or smaller local circuit
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neurons that were postsynaptic to glutamate containing vagal terminals [16]. The local circuit neurons in turn, formed direct synapses with the larger A2 neurons found in the NTS. In this study, both types of noradrenergic neurons exhibited excitatory responses following electrical stimulation of afferent vagal fibers. Considered together, these individual findings suggest that the norepinephrinecontaining neurons of the NTS that have terminal processes innervating structures such as the amygdala may be activated by possibly two mechanisms. One may involve direct activation of these projection neurons by glutamate released from vagal afferents or they may be activated by norepinephrine secreted from local circuit neurons that are interspersed between vagal afferents and the A2 neurons. The likelihood that the first mechanism is involved in the memory storage process was recently confirmed with both behavioral and neurochemical findings [21]. However, there is less evidence indicating whether the norepinephrine elevation in the NTS observed following peripheral physiological arousal or subsequent to glutamate release from vagal afferents is an important component of the processes in which emotionally arousing experiences are encoded into memory storage. Thus, the present studies were conducted to address this shortcoming by examining whether noradrenergic activation of the NTS plays a functional role in affecting memory formation. In experiment 1, rats implanted with injection cannula bilaterally in the NTS were given footshock training in an inhibitory avoidance task. Following training, the rats were removed from the apparatus and given a bilateral injection into the NTS of either PBS or the selective a1-noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, or 10.0 mg / 0.5 ml). The consequences of activating NTS neurons which in turn project to and release norepinephrine in limbic structures were assessed 2 days later by measuring latencies to enter the compartment of the apparatus where footshock was administered during training. To assess whether the changes in memory that may be produced by activating the NTS are not limited to an emotionally aversive task, a separate group of animals were trained in an appetitively motivated Y-maze discrimination task. In this experiment, animals were trained to eat five pellets in the left alley and 10 pellets in the right alley of a Y-shaped maze. On the sixth day of training, after the animals consumed all pellets in both alleys and then animal returned to the right alley, a footshock was administered. Following footshock, PBS or phenylephrine (0.5, 1.0, 5.0, or 10.0 mg / 0.5 ml) was microinfused into the NTS. The animals’ capacity to discriminate between the arms of the maze where shock was or was not delivered was examined on retention tests given 24 and 48 h later. The results of demonstrate that a1-nordrenergic receptors in the NTS play an important role in facilitating synaptic transmission in neurons that project to and release norepinephrine in limbic structures that are involved in memory formation.
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2. Methods
2.1. Subjects One-hundred and ten male Sprague–Dawley rats (275– 300 g) obtained from Hilltop Laboratories (Scottesdale, PA, USA) were used in experiments 1 (n575) and 2 (n530). Upon arrival, the animals were individually housed in plastic cages and maintained on a standard 12-h light–dark cycle with lights on at 07:00 h. The animals were adapted to the vivarium for 7 days without being disturbed during which food and water were available ad libitum.
2.2. Surgery In preparation for surgery each rat was given atropine sulfate (0.2 mg / kg, i.p.; Vedco, St. Joseph, MO, USA) to aid in respiration and then anesthetized with sodium pentobarbital (50 mg / kg; Abbott, North Chicago, IL, USA). After anesthetized, a midline scalp incision was made, and 15-mm 25.0-gauge extra thin wall stainless steel guide cannulas (Small Parts, Miami Lakes, FL, USA) were implanted bilaterally 2 mm above the NTS (AP: 213.30; ML; 61.0 from bregma; DV: 25.6) according to the atlas of Paxinos and Watson [23]. The cannulas and two anchoring screws were affixed to the skull with dental cement, and the scalp was closed with autoclips. Stylets (15 mm, 00 insect dissection pins) were inserted into the injection cannulas to maintain cannula patency. Penicillin (0.1 ml, i.m.; Fort Dodge Pharmaceuticals, Fort Dodge, IA, USA) was administered immediately after surgery. The animals were maintained in a temperature-controlled chamber for approximately 1 h after surgery. For the following 7 days of recovery, the animals were not disturbed in the vivarium.
2.3. Training apparatus and procedures 2.3.1. Experiment 1: inhibitory avoidance training In experiment 1, the rats were trained in a trough-shaped two-compartment inhibitory avoidance apparatus (91 cm in length, 21 cm wide at the top and 6.4 cm at the bottom). A metal sliding door (14.5 cm in height) that opened by retracting into the floor separated a brightly illuminated (40 W) compartment made out of white opaque plexiglas (31 cm long) from a darker compartment constructed of stainless steel plates (60 cm long). On the day of training, the rats were transported to the laboratory from the vivarium approximately 1 h before training. During training, each rat was placed in the white compartment of the inhibitory avoidance apparatus facing the sliding door. After the rat turned 1808 away from this position, the
sliding door was lowered into the floor. Once the animal turned and faced the open entrance to the dark compartment, the timer was initiated, and the latency to enter the dark compartment with all four paws was recorded. Once the animal was fully inside the dark compartment, the sliding door was raised and a 0.45-mA footshock was administered through the metal plates for 0.5 s. The rat was then removed from the training apparatus and given an intra-NTS microinfusion of a phosphate-buffered solution (PBS; n515) or 0.5 (n511), 1.0 (n516), 5.0 (n517), or 10.0 mg (n516) of phenylephrine in 0.5 ml. The animals were returned to the vivarium 1 h after training. Forty-seven hours later the animals were returned to the experiment room and left undisturbed for 60 min prior to the retention test. During the retention test, the animal was placed in the illuminated compartment of the apparatus facing away from the sliding door. After turning 1808, the door was lowered, and the latency to enter the dark compartment with all four paws was recorded (maximum of 600 s) and used as an index of retention.
2.3.2. Experiment 2: Y-maze discrimination task In experiment 2, a trough-shaped Y-maze constructed of stainless steel was used to train animals in a discrimination task. The three alleys of the maze were each 49 cm long318.5 cm high. The floor and ceiling were 4 and 19 cm wide, respectively. The floor of each alley was constructed of two stainless steel plates that were separated lengthwise by a 0.5-cm gap. The floor plates and lower half portion of the inner walls of each alley were covered by three removable trough-shaped cardboard panels (Fig. 1A). The left and right alleys were illuminated by a light (40 W) positioned behind translucent white plexiglas at the end wall of each maze arm. Each alley also contained a plastic food well which protruded from the end wall 2 cm above the floor where food pellets were placed. The rats were placed on a weight maintenance schedule for 7–10 days prior to the beginning of training to reduce their body weight by 15%. This target weight was maintained for the duration of the experiment. The first 2 days of Y-maze training consisted of habituation in which all animals freely explored the maze for 5 min. During habituation and the next 5 days of training, the stainless steel floor of each maze alley was covered with the trough-shaped cardboard panels. After 2 days of habituation, the rats underwent one training trial per day for 6 consecutive days. On each trial, the rat was placed in the stem of the Y-maze facing the end wall and given up to 5 min to locate and consume 10 Noyes Formula A food pellets (45 mg each; P.J. Noyes, Lancaster, NH, USA) placed in the food well protruding from the rear wall of the right alley and five pellets placed in the food well in the left alley. A trial ended when all 15 food pellets were consumed. The subject was then left in the maze to explore
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the alleys for an additional 30 s before being returned to its home cage. Digital timers were used to record: (1) the initial latency to enter either alley at the start of training; (2) the number of seconds required to consume all pellets in the right and left alleys after entering; (3) the number of seconds the rats remained in an alley after pellets were consumed; (4) the cumulative time to consume all pellets, from the beginning of a trial; and (5) the time spent in the non-baited stem (start alley) of the Y-maze. On day 6 of training, the cardboard insert covering the floor of the maze was removed from only the right alley to expose the stainless steel footshock plates. As in the previous days of training, each rat was allowed to consume all food pellets placed in both the left and right alleys. However, when the rat returned to the right alley and approached the empty food well, a 0.35-mA footshock was administered for 0.5 s through the stainless steel plates by a shock generator (Model 82406, Lafayette Instruments, Lafayette, IN, USA). Immediately after the footshock, the rat was removed from the maze and randomly assigned to groups that received a bilateral intra-NTS infusion of either PBS (n58) or 0.5 (n56), 1.0 (n56), 5.0 (n56), 10.0 mg (n54) of phenylephrine in 0.5 ml. Memory for footshock delivery in the right alley was assessed on days 7 and 8 under two separate conditions. On the first retention test (day 7), the cardboard insert was returned to the right alley as during the first 5 days of training (Fig. 1A). Thus, the stainless steel plates from which footshock was delivered on day 6 were concealed from the subjects on this retention test. This manipulation provided an assessment of the subjects’ memory of the footshock training in the absence of the most salient contextual cue associated with the footshock (i.e. the footshock plates). For the second retention test, given on day 8, the cardboard panel in the right alley was again removed to expose the stainless steel plates. On this day, the rats were exposed to the same contextual cues that were present on the day of footshock delivery (Fig. 1B). The two different retention tests were expected to reveal whether NTS activation with phenylephrine affects retention under conditions in which memory is weakened by removing salient cues that are linked or associated with an arousing events such as footshock. The tests were also designed to assess whether memory for an emotional experience is facilitated by activation of postsynaptic a1noradrenergic receptors in the NTS under normal testing conditions. The latency to first enter either alley and the number of seconds to consume all the pellets in the right alley served as indices of retention on both tests.
2.4. Microinjection procedures Immediately after training with footshock in either the inhibitory avoidance or Y-maze discrimination task, each animal was removed from the apparatus and gently restrained by hand in the experimenter’s lap. The stylets were removed and 17-mm 30-gauge injection needles were inserted through the injection cannulas into the NTS. The tips of the injection needles extended 2 mm beyond the base of cannulas at the NTS. The needles were connected to 10 ml Hamilton syringes via PE-20 polyethylene tubing. An automated syringe pump (Sage-Orion, Boston, MA, USA) delivered 0.5 ml of PBS (0.105 g of PO 4 NaH 2 H 2 O and 0.029 g of PO 4 Na 2 H in 100 ml of 9% saline) or 0.5, 1.0, 5.0, or 10.0 mg of phenylephrine (Sigma, St. Louis, MO, USA) into the NTS over a period of 60 s. After 0.5 ml of each test compound was delivered, the needles were retained in the cannulas for an additional 60 s to assure delivery of drugs. The stylets were then reinserted into the cannulas, and the animals were returned to their home cages.
2.5. Histology To verify correct placement of injection needle tips and guide cannulas, each animal was anesthetized with a euthanasia solution and perfused intracardially with saline (0.9%) followed by a 10% formalin solution. The brains were stored in formalin (10%) for approximately 7 days, sectioned on a freezing microtome in 40-mm sections, mounted on glass slides, and stained with cresyl violet. The location of the cannulas and injection needle tips were verified by examining enlarged projections of the slides. A photomicrograph of the injection needle tips in the NTS is displayed in Fig. 2.
2.6. Statistical analysis The behavioral measures from the inhibitory avoidance and Y-maze discrimination tasks are expressed as mean latency6standard errors (S.E.). Between-group comparisons for data collected during the inhibitory avoidance retention test were made with a one-way analysis of variance (ANOVA) followed by Fisher’s post-hoc tests. For the Y-maze discrimination task, each measure that served as indices of memory on day 7 and 8 was compared across the groups with ANOVA followed by Fisher’s post-hoc test. Repeated-measure ANOVA was also utilized to compare latencies across the training days (day 6–8) for
Fig. 1. Photographs of the Y-maze discrimination task used in experiment 2. (A) During habituation and days 1–5 of training, all alleys of the maze were covered with cardboard inserts as shown. On each day of training, five pellets were placed in the food cup at the end of the left maze alley and 10 pellets in the right alley. On day 6, the insert in the right alley was removed exposing the metal plate (B). After all 15 pellets were consumed and the animals returned to the right alley, a footshock (0.35 mA) was delivered for 0.5 s. On the retention test given 24 h after footshock training (day 7), the right alley was covered again concealing the cue that was most closely associated with footshock (A). During the second retention test (day 8), the insert was removed again to expose the metal plate from which footshock was delivered on day 6 (B).
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Fig. 2. A Photomicrograph illustrating placement of injection cannulas aimed above the NTS and the site of injection needle tips in the nucleus of the solitary tract (NTS). The shaded area represents the range of needle tip placements in the NTS.
each measure. Values of P,0.05 were considered significant in all tests.
3. Results
3.1. Experiment 1 Experiment 1 examined whether posttraining activation of a1-noradrenergic receptors in the NTS affects later retention performance. During the day of training and the retention test, the latencies to fully enter the dark compartment with all four paws were measured. A one-way ANOVA on the mean latencies to enter the dark compartment during training revealed no significant differences across the separate dose groups, F(4,70)50.84, P50.51. In the retention test however, there was a statistically significant difference in the mean latencies to enter the dark compartment, F(4,70)53.36, P,0.05. Post-hoc Fisher’s tests revealed that animals in the 5.0 and 10.0 mg phenylephrine groups spent a significantly longer period of time in the illuminated white compartment (M5143 and 166 s, respectively) before entering the dark compartment relative to the PBS controls (M559 s; P,0.05 and P, 0.01; see Fig. 3).
3.2. Experiment 2 The findings from experiment 1 indicate that infusion of the a1-noradrenergic agonist phenylephrine into the NTS
dose-dependently improves memory for footshock training. experiment 2 examined whether phenylephrine’s actions in the NTS are also manifested in a different type of learning condition requiring the animals to make a discrimination response. In experiment 2, animals were trained in an appetitively motivated Y-maze discrimination task and administered a control solution or one dose of the a1noradrenergic agonist phenylephrine (0.5, 1.0, 5.0, 10.0 mg) into the NTS. On the first day of retention testing (day 7), the alley where footshock was delivered 24 h earlier (i.e. right alley) was covered with a cardboard insert. Even though this manipulation concealed the most obvious visual cue that was directly associated with the footshock, the performance of animals given a posttraining infusion of the a1noradrenergic agonist into the NTS was significantly different from that of controls on several measures. For example, a one-way ANOVA revealed that the latencies to first enter the right or left alley were significantly different among the separate dose groups, F(4,25)52.94, P,0.05. Post-hoc Fisher’s test indicated that 1.0 mg phenylephrine group took significantly longer to make an alley entry choice than the PBS control group (P,0.01; Fig. 4). Once the animal entered either alley, the length of time required to consume all food pellets at the end wall was measured. The separate drug groups did not differ in the time required to consume all food pellets in the alley where no footshock was given (i.e. left alley), F(4,25)5 1.27, P.0.05. This finding demonstrates that footshock administered in the right alley did not produce a global
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Fig. 3. Experiment 1. Mean (6S.E.) latencies to enter the dark compartment on the day of training (left graph) and during the retention test in an inhibitory avoidance learning task (right graph). All subjects had entrance latencies of less than 5 s on the training day. In contrast, the latencies to enter the dark compartment on the retention test given 48 h later was significantly longer in the groups given a posttraining injection of either 5.0 (n517) or 10.0 (n516) mg / 0.5 ml of phenylephrine in the NTS relative to controls that received PBS (phosphate buffered saline) (n515). * P,0.05, ** P,0.01.
aversion to the entire Y-maze, but only to the alley where this stimulus was delivered. In contrast, there was a significant overall difference in the time taken to consume food pellets in the right alley between the groups, F(4,25)52.85, P,0.05 (data not shown). For this measure, animals given 1.0 and 5.0 mg of phenylephrine into the NTS took significantly longer to eat food pellets than the PBS controls (P,0.05). The cumulative time to consume
all food pellets in the right alley from the start of the retention test was significantly longer in the 1.0 and 5.0 mg phenylephrine groups relative to controls (P,0.05; Fig. 5). Because some animals took a longer time to eat food in the right alley, the length of time they spend in other places of the Y-maze was also measured. Individual comparisons among the drug groups in the total length of time the animals spent in the left ‘safe’ alley during the retention
Fig. 4. Experiment 2. Mean (6S.E.) latencies to enter either the right or left alley of the Y-maze on retention test 1. The group given a posttraining injection of 1.0 mg / 0.5 ml of phenylephrine into the NTS on day 6 took significantly longer to make an entry choice after being placed in the stem of the Y-maze than the PBS control group. * P,0.01.
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Fig. 5. Experiment 2. Mean latencies (6S.E.) to consume all pellets in the right alley on day 6 (footshock training), day 7 (retention test 1 with footshock plates concealed), and day 8 (retention test 2 with footshock plates revealed). This measure represents the cumulative latency to consume all food pellets in the right alley of the Y-maze from the beginning of the trial. On the day of training, all groups consumed the 10 pellets the right alley in less than 31 s. On the retention test given 24 h following footshock and phenylephrine infusion into the NTS, subjects in the 1.0 and 5.0 mg phenylephrine group took significantly longer than the PBS controls to consume all 10 pellets in the right alley. Similarly, on day 8, subjects in the 1.0 mg phenylephrine group took significantly longer to consume the food pellets than those in the control group; * P,0.05.
test yielded significant between-group differences. As displayed in Fig. 6, animals in the 1.0 mg phenylephrine group remained in the left alley for a significantly longer
period of time than controls or the other dose groups (P,0.05). On the second day of retention testing, the animals were
Fig. 6. Experiment 2. Mean (6S.E.) length of time spent in the left ‘safe’ alley of the Y-maze on days 6–8 of training and retention testing. The length of time spent in the left alley while avoiding entry into the right alley was also measured. On the day of training, all animals spent less than 9 s in the left alley. During the first retention test conducted 24 h later, animals in the 1.0 mg / 0.5 ml phenylephrine group remained in the left alley significantly longer than the PBS group. * P,0.05. The difference in the amount of time spent in the left alley on the second day of retention was significant between PBS controls and the 1.0 mg and 5.0 mg phenylephrine groups * P,0.05.
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placed back in the Y-maze with the footshock plates of the right alley exposed. In contrast to the results from retention test 1, the latency to first enter either alley was not significantly different between the drug groups F(4,25)5 0.90, P5.47. However, there was a significant difference between PBS controls and the 1.0 mg phenylephrine group on the time taken to consume all food pellets in the right alley from the beginning of the retention test (P,0.05; Fig. 5). The same figure also displays the differences in this measure between the drug groups across the days of treatment and the two retention tests. As indicated, on the day of treatment, animals in all groups finished consuming food in the right alley at similar latencies (M513–30 s). Forty-eight hours following the footshock and drug treatment, the animals in the control group (M532.3 s) and lowest (0.5 mg; M542.7) and highest (10.0 mg; M562.5 s) phenylephrine dose groups consumed all pellets in the right alley within the same time frame as the day before. A repeated-measure ANOVA showed that the time taken to consume pellets in the right alley in the 1.0 and 5.0 mg groups was significantly longer on the retention test in comparison to the day of training (M5122.7, 98.3 s, respectively; P,0.05). A comparison of the length of time the animals remained in the left alley to avoid the exposed footshock plates in the right alley revealed that animals in the 1.0 and 5.0 mg phenylephrine groups remained there significantly longer during the retention test than those in the PBS group (P,0.05; Fig. 6). The length of time animals in the 1.0 mg phenylephrine groups remained in the non-baited stem of the Y-maze to avoid entry into the right alley was also significantly longer (P,0.05) than the
PBS controls (Fig. 7). The above findings indicate that activation of noradrenergic receptors in the NTS immediately following an emotionally arousing experience enhances later retention performance reflected by either increased avoidance response or decreased consummatory behavior such as feeding.
4. Discussion The findings from the present experiments demonstrate that posttraining activation of a1-noradrenergic receptors in the NTS improves memory under two separate types of learning conditions. In experiment 1, the a1-noradrenergic receptor agonist phenylephrine was administered bilaterally into the NTS following footshock administration in an inhibitory avoidance task. In the retention test given 48 h later, the latencies to enter the dark compartment for the 5.0 or 10.0 mg phenylephrine groups were significantly longer than those of the control group. This result suggests that activation of a1-noradrenergic postsynaptic receptors in the NTS with phenylephrine affects neural processes involved in consolidating memory for emotionally arousing experiences. The data from experiment 2 confirms and extends those from the first study by demonstrating that phenylephrine exerts similar effects on memory in a discrimination learning task. In the food-motivated Y-maze task, the performance of subjects given either 1.0 or 5.0 mg of phenylephrine was statistically different than that of the control groups on several measures used as indices of
Fig. 7. Experiment 2. Mean (6S.E.) length of time spent in the stem of the Y-maze (non-baited alley) on the second day of the retention test. Animals in the 1.0 mg phenylephrine group remained in the stem of the Y-maze significantly longer than the PBS (* P,0.05) controls while avoiding entry into the right alley.
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retention. Moreover, this pattern of responding was observed on both days of the retention test in spite of the changes made to remove contextual cues that were present only during the day of footshock delivery. These present findings expand the current understanding of specific classes of noradrenergic receptors in the NTS that play a critical role in facilitating information and encoding processes that culminates in better retention performance. Previously, simultaneous activation of both a- and bnoradrenergic receptors in the NTS was shown to influence memory storage processes following emotional arousal [5]. The results from the present studies provide evidence that sole activation of a1-noradrenergic receptors in the NTS is sufficient to modulate memory formation processes. The effects on memory produced by activating a1noradrenergic receptors in the NTS were apparent even in the first retention test in experiment 2 when the shock plates in the right alley were covered concealing the contextual cue that was directly related to the arousing footshock experience. Generally, contextual transformations of this kind where important cues that were present during learning are concealed during testing leads to poor retention performance in control subjects [30–32]. However, the present results seem to indicate that the changes in the learning environment did not influence the retention performance of animals administered 1.0 or 5.0 mg of phenylephrine. Animals in the 1.0 mg dose group took significantly longer to make a choice between entering the right or left alley upon being placed in the stem of the Y-maze than did those in the control group (Fig. 4). Even after the animals entered the covered right alley, those given 1.0 or 5.0 mg of phenylephrine took a significantly longer amount of time to approach and consume the food pellets located at the end wall. The prolonged avoidance of the right alley and food pellets can be interpreted as the animals’ exhibition of their memory for where and how the aversive experience took place and indicates that posttraining activation of a1-noradrenergic receptors in the NTS influences the strength in which new memory traces may be encoded. As a consequence, these new traces are less vulnerable to the influences of contextual changes that are distracting and detrimental to retention performance in non-treated animals. Another measure that served to reflect whether learning occurred following footshock delivery involved the shift in the animals’ alley preferences. The mean time spent in the left alley on the treatment day (day 6) was 6.3 s for PBS group and 7.7 s for 1.0 mg phenylephrine group. However, on the retention test (day 7) the mean latency increased to 26.0 s for the 1.0 mg group which was significantly longer than the mean length of time the controls remained (9.8 s) in this ‘safe’ area of the maze 24 h after footshock. If the circumstances surrounding footshock on the treatment day were significant enough to encode into memory, one would assume that upon exposure, the animals would adapt their behavioral response to avoid the footshock thereafter.
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On the second day of retention testing, the cardboard panel was removed in the right alley and the stainless steel footshock plates were exposed. As a result, the latencies to enter and consume food pellets in the right arm were longer than the previous day for all groups. Even though retention performance was more evident in all groups when the metal plates were visible, there were still significant differences between the control and 1.0 and 5.0 mg phenylephrine groups in the time taken to either consume all pellets in the right alley, or in the length of time the animals remained in the left alley or the stem of the Y-maze during the retention test. Thus, the animals in the 1.0 and 5.0 mg phenylephrine groups could discriminate between which alley is ‘safe’ relative to the alley of the maze where an unexpected and unpleasant event was experienced. Considered together with the findings from the first retention test, these patterns of contrasting behavior by the animals in the control and the 1.0 and 5.0 mg phenylephrine groups support a premise that increased noradrenergic neurotransmission in the NTS facilitates neural processes involved in encoding and storing information into memory. The results also indicate that activation of a1-noradrenergic receptors in the NTS is an important component in the mechanisms underlying modulation of emotional, contextual, and stimulus–response associations into memory storage processes. In experiment 2, the doses of phenylephrine that improved memory in the Y-maze discrimination learning task (1.0 and 5.0 mg) were lower than the effective doses in experiment 1 (5.0 and 10.0 mg). There are several possibilities for this finding. First, animals trained in the Y-maze task were on a food-restricted schedule for approximately 2 weeks prior to administration of the drugs into the NTS. Although the food deprivation procedure used to reduce weight by 15% does not alter the sensitivity to shock or acquisition of the avoidance response [20], it does elevate circulating concentrations of the hormone epinephrine in plasma [11]. Since a1-noradrenergic receptor activation in the NTS is one mechanism by which physiological signals from the periphery are transmitted to memory-encoding structures in the brain following emotional arousal, a lower dose of phenylephrine might be sufficient to produce an optimum memory enhancement with higher levels of epinephrine already circulating in the periphery. Another possible reason for a lower dose being effective in experiment 2 could have been the training procedure. In the inhibitory avoidance task, the footshock was administered in a novel training environment. In contrast, in the food-motivated discrimination task the animals were placed in the Y-maze for at least 60 s on each day of training for 5 days prior to the footshock. Since the aversive stimulus was never experienced during this period and only encountered on the day of treatment, it is possible that the animals associated the footshock with the context of right alley much more substantially with an element of surprise and elicited more arousal, and only a lower dose
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of phenylephrine was required to enhance memory retention in the task. The effects of phenylephrine infusion into the NTS on memory demonstrated in the present study are attributed to activation of a1-noradrenergic receptors localized on noradrenergic neurons in the NTS [8,15,17,13,14,28]. aand b-noradrenergic receptors in the NTS are likely to be activated by norepinephrine released from interneurons between vagal afferents and NTS neurons [16] or from noradrenergic neurons in the area postrema which also receives vagal input [2,4]. Therefore, an input to trigger norepinephrine release and receptor activation in the NTS could originate from signals carried by the vagus nerve arriving from the periphery regarding the physiological state of an organism. The afferent branches of the vagus nerve innervate a variety of visceral organs (for review, see Ref. [12]) and carry various types of signals including cardiovascular, respiratory, gastrointestinal, endocrine, autonomic, and immune to the brain via the NTS [1]. Therefore, information regarding changes in physiological state elicited by emotional arousal may be conveyed by the vagus nerve and generate alterations in the noradrenergic system in the NTS. In fact, an increase in blood pressure, one physiological response that may accompany emotional arousal, has been shown to elevate extracellular norepinephrine levels within the NTS [6]. Norepinephrine released in the NTS in response to peripheral activation stimulates NTS noradrenergic neurons that project to forebrain structures [26]. One of its termination sites is the amygdala [34,7,24], a limbic structure implicated in modulation of memory storage [3,19]. Norepinephrine release in the amygdala has been extensively established as a neural substrate for memory modulation elicited by peripheral arousal [9,33]. The results from the present study indicate that the A2 noradrenergic neurons in the NTS contribute to memory processing for emotionally arousing events. More specifically, catecholamines in the NTS and the activation of A2 neurons play an important role in insuring that the consequences of emotionally significant events are made more salient and encoded in the brain.
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