Lesions of the magnocellularis induced by 192 IgG-saporin block memory enhancement with posttraining in the basolateral

Ann E. Power*†, Leon J. Thal‡§, and James L. McGaugh*

*Center for the Neurobiology of Learning and Memory, and Department of Neurobiology and Behavior, University of California, Irvine, CA 92697-3800; ‡Department of Neurosciences, University of California, San Diego, CA 92093; and §Department of Neurology, Veterans Administration Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161

Contributed by James L. McGaugh, November 26, 2001 Extensive evidence indicates that drugs and stress hormones act in Extensive evidence indicates that the BLA mediates the the basolateral amygdala (BLA) to modulate memory consolida- modulation of memory traces that are processed and͞or stored tion. The BLA projects to the nucleus basalis magnocellularis in other brain regions (40) such as the (41), the (NBM), which sends broad projections to the . (11, 37, 41) and the entorhinal cortex (unpublished NBM-cortex projections have been implicated in learning, memory findings). A possible mode of influence for the BLA on memory storage, and plasticity. The current study investigated whether the storage processes in other brain regions may be its excitatory cholinergic NBM-cortex projections are involved in BLA-mediated projection to the cholinergic cells in the NBM (42, 43). The modulation of memory consolidation. Bilateral cholinergic cell cholinergic cells in the NBM project widely to neocortex (44). lesions of the NBM were induced in rats with infusions of 192 This projection is strongly implicated in cortical plasticity, IgG-saporin (0.1 ␮g͞0.5 ␮l per side). Additionally, cannulae were learning, and memory (45–47). Electrical stimulation of the implanted bilaterally in the BLA. One week after surgery, the rats amygdala induces low-voltage fast activity in the neocortex that were trained in an inhibitory avoidance task and, immediately is blocked by cholinergic antagonism in the neocortex (48) or after training, norepinephrine (0.3 ␮g, 1.0 ␮g, or 3.0 ␮gin0.2␮l) lidocaine inactivation of the NBM (49). Furthermore, stimula- or vehicle (PBS) was infused bilaterally into the BLA. Norepineph- tion of the amygdala potentiates the cortical response to so- rine infusions produced a dose-dependent enhancement of 48-h matosensory stimulation or NBM stimulation (50, 51). retention (0.3 ␮g and 1.0 ␮g doses enhanced) in nonlesioned rats The current experiment investigated whether the NBM-cortex but did not affect retention in NBM-lesioned rats. Choline acetyl- projections are involved in memory modulatory processes me- transferase assays of frontal and occipital cortices confirmed the diated by the BLA. Rats were given bilateral NBM-lesions with NBM lesions. These findings indicate that cholinergic NBM-cortex the p75 NGF receptor-mediated immunotoxin 192 IgG-saporin projections are required for BLA-mediated modulation of memory or sham infusions and implanted with bilateral cannulae aimed consolidation. at the BLA. Eight days after surgery, rats were trained in the IA task and given intra-BLA infusions of norepinephrine or vehicle ͉ inhibitory avoidance ͉ memory modulation ͉ rat immediately after training. If the NBM-cortex pathway is in- volved in BLA-mediated memory-modulatory processes, then ystemic administration of drugs, including adrenergic ago- the lesions should interfere with norepinephrine-induced mem- Snists (1–3) and GABAergic antagonists (4–6), given imme- ory enhancement as assessed by performance on a 48 h retention diately after training, enhances memory for several kinds of test. training. The basolateral amygdala (BLA) (4, 8–12) and its major efferent pathway, the (13–16), are critically Methods involved in the modulation of memory consolidation induced by Subjects. Male Sprague–Dawley rats (Charles River Breeding posttraining pharmacological treatments. In addition, consider- Laboratories) weighing Ϸ300 g at the time of surgery were used able evidence indicates that memory modulation by systemic for this experiment. The rats were individually housed in a drugs requires ␤-adrenergic activation in the BLA (17–21). temperature-controlled (22°C) vivarium on a 12-hr light͞12-hr Inhibitory avoidance (IA) tasks have been used most exten- dark cycle, with food and water freely available, and were sively in memory modulatory studies, as the use of a single allowed 1 week to habituate to the vivarium before surgery. training trial enables selective modulation of the consolidation phase of memory. Memory for IA training can be modulated by Surgery. The rats were anesthetized with sodium pentobarbitol posttraining intra-BLA infusions of adrenergic (21–25), (50 mg͞kg i.p.; Sigma) and were given atropine sulfate (0.1 mg, GABAergic (6, 26), opioid peptidergic (19, 27, 28, 87), glucocor- i.p.) to maintain respiration and sterile saline (2.5 ml, s.c.) to ticoid (21, 30, 31), or cholinergic drugs (32–35). As with systemic prevent dehydration. Each rat was placed in a small-animal treatments, memory modulation induced by posttraining intra- stereotaxic frame (Kopf Instruments, Tujunga, CA) and given BLA drug infusions acting on glucocorticoid, GABA, or opioid bilateral infusions of 192 IgG-saporin solution (0.1 ␮gin0.5␮l receptors requires concurrent ␤-adrenergic activation in the BLA (19, 21, 36, 37). Furthermore, it was shown recently in our laboratory that levels of amygdalar norepinephrine assessed Abbreviations: BLA, basolateral amygdala; IA, inhibitory avoidance; NBM, nucleus basalis after training predict long-term retention of IA (unpublished magnocellularis; ChAT, choline acetyltransferase. findings). The adrenergic-dependent memory modulation fur- †To whom reprint requests should be addressed at: 218 BRL, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92697-3800. E-mail: ther depends upon muscarinic cholinergic activation in the BLA [email protected]. (31–33, 38, 39), which likely originates from the major amyg- The publication costs of this article were defrayed in part by page charge payment. This

dalopetal projection of the nucleus basalis magnocellularis article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. NEUROBIOLOGY (NBM) (37). §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.022627799 PNAS ͉ February 19, 2002 ͉ vol. 99 ͉ no. 4 ͉ 2315–2319 Downloaded by guest on September 27, 2021 of 0.1 M PBS; Chemicon) or sham infusions into the NBM region tein content of the homogenates was determined by the method of the , according to the following coor- of Lowry et al. (56) and expressed in nmol of acetylcholine dinates based on the atlas of Paxinos and Watson (52): 1.4 mm formed per h per mg of protein. Data from lesioned rats whose posterior and 2.7 mm lateral to bregma, 7.8 mm below the dura. ChAT levels were within the range of the controls were excluded. The dose and infusion volume of 192 IgG-saporin chosen for this experiment were based on a dose-response study of intraparen- Histology. After the cortical dissection for biochemical analysis, chymal NBM-infusions of 192 IgG-saporin (53). Each rat then the brains were fixed in 4% (wt͞vol) formaldehyde solution for was placed in a second stereotaxic frame and implanted with 2 days. Brains then were stored in 30% (wt͞vol) sucrose in saline bilateral cannulae (15 mm, 23 gauge) aimed at the BLA. The solution until slicing. Slices (40 ␮m) including the NBM and the cannulae were positioned Ϸ2 mm above the BLA with the BLA were collected on a freezing microtome and stained with following coordinates: 2.8 mm posterior and 5 mm lateral to thionine. Stained sections were analyzed with a light microscope bregma, 6.5 mm below the skull surface (53). Dental cement to determine the infusion sites. Rats that did not receive both fixed the cannulae and two anchoring surgical screws to the skull. posttraining infusions within the BLA were excluded from the Stylets (15-mm long 00 insect-dissection pins) then were inserted data analysis. into the cannulae to maintain patency. The rat was retained in an incubator until it recovered from the anesthesia. Rats were Statistical Analysis. As none of the rats included in the behavioral allowed 1 week to recover from the surgery before behavioral data analysis after the histological and biochemical analyses had training and were handled for 1 min on each of the 4 days floor (0 s) or ceiling (600 s) retention latencies, parametric tests preceding training to habituate them to the infusion procedure. were used to detect statistically significant treatment effects. The behavioral data were analyzed with a two-way ANOVA with IA Task. The IA apparatus is a trough-shaped alley divided by a NBM treatment (two levels, sham or lesion) and BLA treatment sliding door into two compartments, a lit safe compartment (31 (four levels, vehicle and three doses of norepinephrine) as the cm long) and a darkened shock compartment (60 cm long). between-subject variables. To confirm that the 192 IgG-saporin During training, each rat was placed into the lit safe compart- lesion did not block all IA learning, the overall mean training ment facing away from the dark compartment, with the sliding latencies were compared with the mean testing latencies of door open. The rat was allowed to enter the darkened shock vehicle-infused groups in a two-way ANOVA with session as the compartment. After each rat completely entered the dark com- variable (two levels, training or testing). The ChAT assay data partment with all four paws, the sliding door was closed, and the for each sample area (cortical region, side) were analyzed with animal received a mild inescapable foot shock (0.5 mA, 1.0 s) and a two-way ANOVA with NBM treatment (two levels, sham or remained in the dark compartment for 15 s after the shock. The lesion) and cortical region (four levels, left and right frontal, left rat then was given the appropriate infusions into the BLA. and right occipital) as the between-subject variable. Fisher’s post Forty-eight hours after IA training, retention for the training hoc tests were used to detect the sources of the significances was tested. During testing, each rat was placed in the lit safe detected by the ANOVAs. In all cases, P values of less than 5% compartment, as it was in training. The latency for the rat to (0.05) were the criteria for significant differences. The number enter the dark shock compartment was recorded. The maximum of animals per group are reported in the figure legends. latency cutoff was 600 s. Better retention was inferred from longer latencies to enter the shock compartment during the Results retention test. Histology. The locations of the posttraining infusion sites are shown in Fig. 1A. A representative example of an infusion needle Drug Administration. Immediately after IA training, rats received track terminating within the BLA, per the criteria of this bilateral infusions (0.2 ␮l per side) of (ϩ͞Ϫ)-norepinephrine experiment, is shown in Fig. 1B. Data from 13 rats whose HCl (Sigma) or vehicle (0.1 M PBS) into the BLA. This infusion needle tracks terminated outside the BLA were ex- conservative volume has previously been shown to provide cluded from analysis. selective infusion into the BLA (6, 30). The doses of norepi- nephrine (0.3 ␮g, 1.0 ␮g, and 3.0 ␮g) were chosen on the basis Biochemistry: Choline Acetyltransferase Activity. As summarized in of unpublished dose-response data from this laboratory and, as Table 1, the 192 IgG-saporin lesions of the NBM significantly ϭ Ͻ expected, were of slightly higher concentrations than those of reduced cortical ChAT activity (F1,300 583.05, P 0.0001). (Ϫ)-norepinephrine HCl (discontinued) used in previous studies Significantly less ChAT activity was detected in the left and right (17, 54). Solutions were infused over 25 s into the BLA by frontal cortical regions of lesioned animals than in the left and ϭ Ͻ automated syringe pumps (Sage Instruments, Boston) by means right occipital regions (region effect: F3,300 14.41, P 0.0001; ␮ ϫ ϭ Ͻ of 30-gauge injection needles (17 mm) attached to 10- l Ham- region NBM treatment effect: F3,300 65.45, P 0.0001). The ilton microsyringes (by polyethylene tubing, PE-20). Needles left and right hemisphere ChAT activity levels did not differ were left in place for an additional 30 s after the infusions to significantly from each other in either cortical area (P Ͼ 0.05). allow diffusion into the BLA. After drug infusions, the stylets The lesions produced a pronounced ChAT reduction in the were replaced into the cannulae, and the rats were replaced in frontal cortical regions relative to the respective ChAT activity their home cages. levels of sham animals (P Ͻ 0.0001). The mean ChAT levels of the left and right frontal cortices of the lesioned rats were 67.0% Biochemistry: Choline Acetyltransferase Activity. One day after the and 63.5% less than the mean ChAT level of the controls, IA retention test, rats were killed by decapitation. The brains respectively. The lesions also produced a reduction of ChAT in were rapidly removed, chilled in saline solution (0.9%) and the occipital cortical regions relative to the respective ChAT dissected on ice. Frontal and occipital cortex samples were activity levels of sham animals (P Ͻ 0.0001). The mean ChAT collected from each hemisphere of all rats. Parietal cortex levels of the left and right occipital cortices of the lesioned rats regions were taken with the histological blocks for confirmation were 19.8% and 20.4% less than the mean ChAT level observed of infusions sites (described below) and were, therefore, unavail- in the controls, respectively. Data from five rats were excluded able for biochemical processing. Samples were kept frozen from the behavioral analysis because of insufficient ChAT (Ϫ70°C) until analyzed. Each sample was sonicated in 400 ␮lof reduction. Only ChAT activity levels of rats included in the 50 mM PBS (pH 7.4) and processed for the assay of choline behavioral analysis are included in the ChAT activity summary acetyltransferase (ChAT) by the method of Fonnum (55). Pro- table.

2316 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.022627799 Power et al. Downloaded by guest on September 27, 2021 Table 1. Activity of choline acetyltransferase Activity (nmol Ach͞h͞mg protein)

Cortical region Control 192 IgG-saporin Difference, %

Frontal, left 33.20 Ϯ 0.84 10.98 Ϯ 0.86 67.0 decrease* Frontal, right 32.37 Ϯ 0.77 11.51 Ϯ 1.08 64.5 decrease* Occipital, left 28.36 Ϯ 0.53 22.75 Ϯ 0.72 19.8 decrease* Occipital, right 28.83 Ϯ 0.68 22.95 Ϯ 0.85 20.4 decrease*

*Sham vs. 192 IgG-saporin lesion ChAT activity, significance P Ͻ .0001, means Ϯ SEM.

cantly longer latencies to enter the shock compartment on the Ϯ retention test than the mean training latency (14.17 1.03; F1,108 ϭ 6.49, P Ͻ 0.05), indicating that the lesion did not block all IA learning. Fisher’s post hoc tests indicated that the 0.3 ␮g(P Ͻ 0.01) and 1.0 ␮g(P Ͻ 0.001) doses of norepinephrine signifi- cantly enhanced retention in the sham groups (121.2 Ϯ 35.0 s and 167.9 Ϯ 57.8 s, respectively) compared with the vehicle-infused sham group (Fig. 2). However, the mean retention latencies (17.4 Ϯ 3.0 s and 22.9 Ϯ 4.1 s, respectively) for the lesioned groups that received these doses of norepinephrine (which were memory enhancing in sham rats) did not differ from those of the vehicle infused lesioned group (P Ͼ 0.05). The mean retention latencies of the 3.0 ␮g dose norepinephrine groups (sham, 54.0 Ϯ

Fig. 1. Only data from animals with posttraining infusion sites within the ᭜ BLA were included in the analysis. (A) represents the infusion needle tip loci Fig. 2. Intra-BLA infusions of norepinephrine immediately after IA training for a random sample of 30 rats included in the behavioral analysis. Coordinates produce a dose-dependent enhancement of 48-h retention in sham-operated indicate distance posterior to bregma. (B) Photomicrograph of a representa- rats. The gray bar (tr.) indicates the mean training latency across all groups, tive example of an infusion needle terminating within the BLA. which did not differ between any of the groups (P Ͼ 0.05). The mean training latency was significantly less than the retention latencies of the sham and lesion vehicle-infused groups (❖, P Ͻ 0.05), which did not differ from each Inhibitory Avoidance. An ANOVA (n ϭ 77) revealed a significant other (P Ͼ 0.05). An ANOVA revealed significant main effects of the norepi- ϭ Ͻ nephrine infusions, the 192 IgG-saporin lesion, and a significant norepineph- effect of the 192 IgG-saporin NBM-lesion (F1,69 12.82, P rine infusion ϫ lesion interaction effect (P Ͻ 0.05). The 0.3 ␮g(*, P Ͻ 0.01) and 0.001), a significant effect of the intra-BLA norepinephrine ␮ Ͻ ϭ Ͻ the 1.0 g(**, P 0.001) doses significantly enhanced retention relative to the infusions (F3,69 2.83, P 0.05) and a significant interaction of vehicle-infused sham group. Rats with the 192 IgG-saporin NBM-lesions did the lesion and the BLA-norepinephrine treatment on IA reten- not show memory enhancement with norepinephrine infusions. The mean ϭ Ͻ ␮ Ͻ ␮ Ͻ tion (F3,69 3.02, P 0.05). The 192 IgG-saporin NBM-lesions retention latencies of the 0.3 g (#, P 0.05) and the 1.0 g (##, P 0.001) dose did not affect training latencies in any group or retention lesion groups were significantly shorter than those of their respective dose- latencies in vehicle-infused groups. That is, vehicle-treated le- matched sham control groups, but did not differ from that of the vehicle- Ϯ infused sham or lesion groups (P Ͼ 0.05). (The number of animals per group sioned rats (20.7 3.8 s) performed like vehicle-treated sham ␮ Ϯ Ͼ are as follows: sham-vehicle, 11; sham-norepinephrine 0.3 g, 10; sham- rats (21.9 6.3 s; P 0.05) on the retention test, and there was norepinephrine 1.0 ␮g, 10; sham-norepinephrine 3.0 ␮g, 9; lesion-vehicle, 9;

ϭ Ͼ NEUROBIOLOGY no effect of the NBM lesion on training latency (F1,69 0.40, P lesion-norepinephrine 0.3 ␮g, 7; lesion-norepinephrine 1.0 ␮g, 8; lesion- 0.05). The vehicle-infused sham and lesion groups had signifi- norepinephrine 3.0 ␮g, 14.)

Power et al. PNAS ͉ February 19, 2002 ͉ vol. 99 ͉ no. 4 ͉ 2317 Downloaded by guest on September 27, 2021 30.4; lesion, 28.0 Ϯ 4.6 s) did not differ from those of their the BLA, and that long-term memory of IA training involves respective controls (P Ͼ 0.05). entorhinal and posterior parietal cortices (81–83). Because the BLA sends and receives projections to and from Discussion both the NBM and the cortex (42, 44, 84, 85), there are several The present study investigated whether the cholinergic cells in plausible mechanisms by which the NBM-cortex projections may the NBM projecting to cortex are involved in BLA-mediated be involved in mediating BLA-driven memory modulation. The modulation of memory. ChAT assays confirmed the reduction of mechanism of cholinergic NBM-neocortical projection involve- cholinergic input to the neocortex induced by infusion of the ment in BLA-mediated memory modulation may simply involve immunotoxin 192 IgG-saporin into the NBM. Norepinephrine direct excitation of the NBM by the BLA; that is, BLA activation infusions produced a dose-dependent enhancement of retention may directly facilitate the NBM-cortex projections and thereby in sham rats but did not enhance retention in NBM-lesioned rats. enhance memory storage processes in the cortex. Evidence that The finding that posttraining intra-BLA norepinephrine in- stimulation of the BLA produces scopolamine-sensitive, NBM- fusions enhanced IA retention in nonlesioned controls is con- dependent neocortical desynchronization (48, 49) and potenti- sistent with those of numerous reports (14, 17, 18, 22, 24, 25, 54, ates cortical responsivity to somatosensory stimulation or NBM 57, 58). Furthermore, in vivo microdialysis-HPLC studies have stimulation is consistent with this view (50, 51). Also, it is demonstrated that systemic injections of epinephrine or a foot- possible that the BLA-cortex projections may directly influence shock induce norepinephrine release in the amygdala (59), and the cortex in a manner that requires coincident cholinergic that norepinephrine release in the amygdala during IA training activation in the cortex from the NBM to modulate memory storage. Alternatively, it may be that direct cortical input to the correlates highly with performance on a 48-h retention test BLA is influenced by the level of cholinergic activation the (unpublished findings). This convergence of evidence from cortex receives from the NBM. Finally, it may be that a combi- behavioral pharmacological and in vivo microdialysis-HPLC nation of these mechanisms is involved. It is worth noting that studies strongly suggests that modulation of memory consolida- ␤ 192 IgG-saporin selectively lesions corticopetal cholinergic neu- tion is driven by norepinephrine acting on -adrenergic recep- rons, as the amygdalopetal , which may provide impor- tors in the BLA (60). tant input to the BLA during memory-modulatory processes The current finding that the intraparenchymal 192 IgG- (35), do not express the p75 low-affinity NGF receptor to which saporin lesions of the NBM did not produce a deficit in IA this immunotoxin binds (64, 65, 76). retention is consistent with previous findings that impairment on The BLA receives projections from many diffusely projecting this task is produced by intracerebroventricular (icv) adminis- Ͼ systems associated with stress and arousal, including noradren- tration of saporin that produces 90% ChAT reduction in the ergic (86), dopaminergic (7), and cholinergic projections (44, medial septum and in combination 85). Thus, the present findings suggest that memory impairment Ͼ with 80% ChAT reduction in the NBM (61). Generally, in animals with immunotoxic lesions of the NBM may depend on memory deficits are only consistently produced with icv cholin- the magnitude of stress and͞or arousal associated with the task, ergic lesions that disrupt Ͼ 75–85% of ChAT and, therefore, the extent to which the BLA is activated during activity of the total cholinergic basal forebrain (62–64). With an memory consolidation. This possible role for the cholinergic experiment using a training protocol similar to that of the NBM-cortex projection during memory consolidation is not in current study, Wenk et al. (65) found no deficits on several conflict with the hypothesis that it may be involved in attentional measures of IA, including 48-h retention latency and freezing processes during learning (29, 62, 65, 76). behavior in rats with saporin lesions of the NBM. However, In summary, the present findings demonstrate a role for the Torres et al. (66) found a small but significant impairment on IA NBM-cortex projections in BLA-driven memory modulatory retention with saporin NBM lesions. The relative ineffectiveness processes. 192 IgG-saporin lesions of the NBM blocked memory of immunotoxic NBM lesions to impair memory relative to enhancement by posttraining intra-BLA infusion of norepineph- excitotoxic NBM lesions suggests that the robust memory deficits rine in an IA task. These data are consistent with previous produced with excitotoxic lesions (67–70) may be caused by findings in suggesting that the BLA influences memory storage damaged amygdalopetal cholinergic projections (35, 71, 72) in other brain areas. These findings further suggest that BLA and͞or damaged noncholinergic cells (63, 73–76). activation may modulate memory-storage processes in the neo- The present finding is that 192 IgG-saporin NBM lesions, cortex by means of a mechanism that requires activation of the which did not produce IA impairment, blocked memory en- NBM-cortex projections. hancement induced by norepinephrine infusions administered into the BLA. This finding strongly suggests that the NBM-cortex The authors thank Andrew Chen for processing ChAT assays, Anna projections may mediate BLA-driven modulation of memory Litmanovich and ThuyVu Ho for valuable technical assistance, and Norman M. Weinberger for providing the 192 IgG-saporin, and Jan storage or processing in the neocortex. Moreover, this finding is Bures and Paul Gold for their helpful comments on a previous draft. This consistent with previous reports in indicating that the BLA study was supported by National Institutes of Mental Health Grant modulates memory storage in other brain regions (14, 16, 54, MH12526 (to J.L.M.) and National Institutes of Health Grant AG17533 77–80) rather than influencing consolidation of memories within (to L.J.T.).

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