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. Author manuscript; available in PMC: 2025 Nov 1.
Published in final edited form as: Neurobiol Learn Mem. 2024 Oct 5;215:107988. doi: 10.1016/j.nlm.2024.107988

Basolateral amygdala inputs to the nucleus accumbens shell modulate the consolidation of cued-response and inhibitory avoidance learning

Bess Glickman a,*,1, Krista L Wahlstrom b,1, Jason J Radley a,b,c, Ryan T LaLumiere a,b,c
PMCID: PMC11536963  NIHMSID: NIHMS2029352  PMID: 39369810

Abstract

The basolateral amygdala (BLA) modulates different types of memory consolidation via distinct projections to downstream brain regions in multiple memory systems. Prior studies indicate that the BLA projects to the nucleus accumbens shell (NAshell) and that these regions interact to influence some types of behavior. Moreover, previous pharmacological work suggests the BLA and NAshell interact to influence memory. However, the precise role of the BLA-NAshell pathway has never been directly investigated in the consolidation of different types of memory including cued-response, spatial, or inhibitory avoidance (IA) learning. To address this, male and female Sprague-Dawley rats received optogenetic manipulations of the BLA or BLA-NAshell pathway immediately following training in different learning tasks. An initial experiment found that optogenetically inhibiting the BLA itself immediately after training impaired cued-response retention in a Barnes maze task in males and females, confirming earlier pharmacological work in males alone. Subsequent experiments found that BLA-NAshell pathway inhibition impaired retention of cued-response and IA learning but had no effect on retention of spatial learning. However, the present work did not observe any effects of pathway stimulation immediately after cued-response or IA learning. Together, the present findings suggest the BLA modulates the consolidation of cued-response and IA, but not spatial, memory consolidation via NAshell projections.

Keywords: Cued-response, Inhibitory avoidance, Memory, Nucleus accumbens shell, Basolateral amygdala, Optogenetics

1. Introduction

The basolateral amygdala (BLA) modulates the consolidation of many different kinds of memories through interactions with a variety of downstream brain regions (Hatfield & McGaugh, 1999; Huff et al., 2013; LaLumiere, Pizano, & McGaugh, 2004; Packard et al., 1994), and this ability of the BLA is likely mediated via distinct efferent projections (McGaugh, 2002; McIntyre et al., 2012). For example, recent findings from our laboratory suggest that BLA projections to the ventral hippocampus influence the consolidation of footshock, but not context, learning (Huff et al., 2016), whereas projections to the medial entorhinal cortex (mEC) influence the consolidation of spatial/contextual memories (Wahlstrom et al., 2018). Previous work indicates that the BLA also projects to the nucleus accumbens shell (NAshell) (Brog et al., 1993; Christie et al., 1987; Kelley et al., 1982) and that these two regions interact to influence some types of behavior (Britt et al., 2012; Millan et al., 2017). However, prior research has not investigated the role of BLA-NAshell projections in different types of memory consolidation.

Multiple memory systems in the brain process the consolidation, storage, and expression of distinct types of memories (Kesner et al., 1993; McDonald & White, 1993; Packard & McGaugh, 1996; Poldrack & Packard, 2003). Previous research indicates that the BLA interacts with these different systems to modulate the consolidation of cued-response, inhibitory avoidance (IA), and spatial memory consolidation (Liang & McGaugh, 1983a, 1983b; Liang et al., 1990; Packard et al., 1994). In addition to relying on different systems in the brain, these tasks involve different types of learning with different valences. The IA task is an aversive learning and memory task where a rat learns to associate a specific context with a footshock (Malin & McGaugh, 2006). In contrast, cued-response and spatial learning tasks using a Barnes maze may have both rewarding and aversive components. The Barnes maze allows for probing of similar types of memory as the Morris water maze yet is likely less physically demanding and less stressful as there is not a swimming component (Gawel et al., 2019; Paul et al., 2009). In these Barnes maze tasks, rats learn to find an escape port by either using extramaze cues (spatial task) or an intramaze cue (cued-response task) (Wahlstrom et al., 2020; Wahlstrom et al., 2018). Although finding the escape port may be rewarding, the initial motivation to find it depends on the aversiveness of being on an open, exposed platform. Whether the BLA-NAshell pathway influences consolidation across these different types of learning is unknown. Indeed, there is a lack of consensus in recent research about whether distinct BLA-NA projections modulate behaviors of opposing valence (Beyeler et al., 2018; Kim et al., 2016; Zhang et al., 2021), raising the question of whether the BLA-NA pathway differentially modulates these types of learning.

Thus, the present work examined the BLA-medial NAshell pathway as a potential circuit-based mechanism by which the BLA modulates the consolidation of cued-response, IA, and spatial learning to determine whether this pathway modulates memory consolidation in a task-dependent manner. To address this, rats received optogenetic manipulation of this pathway immediately following cued-response or spatial training in a Barnes maze, as we have done previously (Wahlstrom et al., 2020; Wahlstrom et al., 2018), or immediately following IA training. An initial experiment found that optogenetically inhibiting BLA cell bodies immediately after cued-response training impaired retention, confirming earlier pharmacological work in males alone (Packard et al., 1994). Furthermore, the results reveal that posttraining BLA-NAshell inhibition impaired retention for cued-response and IA learning with no effect on spatial learning. However, posttraining stimulation of the same pathway had no effect on cued-response or IA retention. Together, these findings suggest a circuit-based mechanism by which the BLA influences both cued-response and IA memory consolidation.

2. Materials and Methods

2.1. Subjects

Male and female Sprague-Dawley rats (185–200 g and 150–175 g, respectively, at time of first surgery; Envigo; n = 217 included in the final analyses) were used for this study. All rats were single housed in a temperature-controlled environment under a 12 h light/dark cycle (lights on at 07:00) and allowed to acclimate to the vivarium at least 2 days before surgery. Food and water were available ad libitum throughout all training and testing. All procedures were in compliance with the National Institutes of Health guidelines for care of laboratory animals and were approved by the University of Iowa Institutional Animal Care and Use Committee.

2.2. Surgery

Rats were anesthetized with 3–4 % isoflurane before receiving pre-emptive analgesia (2 mg/kg meloxicam) and were then placed in a stereotaxic apparatus (Kopf Instruments). Surgical anesthesia was maintained with 2–3 % isoflurane. All rats received virus microinjections (0.35 μL; rAAV5-CaMKIIα-eNpHR3.0-eYFP (inhibitory opsin), rAAV5-CaMKIIα-hChR2(E123A)-eYFP (excitatory opsin), or rAAV5-CaMKIIα-eYFP (no-opsin control); University of North Carolina Vector Core) delivered bilaterally through a 33-gauge needle into the BLA (males: 2.6 mm posterior and 4.65 mm lateral to bregma and 7.8 mm ventral to skull surface; females: 2.2 mm posterior and 4.4 mm lateral to bregma and 7.4 mm ventral to skull surface). Histological analysis indicated transduction of neurons throughout the basolateral complex of the amygdala, including the lateral nucleus. Thus, the present manuscript refers to the entire transduced region as the “BLA” (Wahlstrom et al., 2020; Wahlstrom et al., 2018). Rats underwent a second surgery two (Experiment 1) or four weeks (Experiments 2–4) later, allowing sufficient time for robust opsin expression in BLA cell bodies and axon terminals, respectively. Optical probes were aimed bilaterally at the BLA (Experiment 1) (males: 2.8 mm posterior and 5.1 mm lateral to bregma and 7.1 mm ventral to skull surface; females: 2.8 mm posterior and 5.0 mm lateral to bregma and 7.1 mm ventral to skull surface) or the medial NAshell (Experiments 2–4) (males: 1.3 mm anterior and 2.1 mm lateral to bregma and 7.2 mm ventral to skull surface (10° angle); females: 1.3 mm anterior and 2.1 mm lateral to bregma and 6.8 mm ventral to skull surface (10° angle)) and secured by surgical screws and dental acrylic. Fiber optic placement was angled to provide maximal illumination of the entire NAshell (Millan et al., 2017). For each rat, a single cannula (Plastics One) that did not penetrate the skull was secured in the dental acrylic to serve as an anchor point for connection to optic leashes during optical illumination to reduce tension on the optical fiber connections. The rats recovered for one week before behavioral training.

2.3. Optical manipulations

Optical probes were constructed by gluing an optical fiber (200 μm core, multimode, 0.50NA) into a metal ferrule (length: 7.95 + 8.00 mm, bore: 230 – 240 μm, concentricity: <20 μm). The fiber extended beyond the ferrule end for implantation into tissue. The other end of the optical probe was polished and, during light delivery, connected to another optical fiber via a ceramic split sleeve. This optical fiber was threaded through a metal leash to protect the fiber from being damaged by the rat and attached to a 1:2 splitter (FC/PC connection) to permit bilateral illumination. The splitter’s single end was attached to an optical commutator (Doric Lenses) allowing free rotation of the optic leash connected to the rat. A fiber patch cable connected the commutator to the laser source (DPSS, 300 mW, 561 nm for eNpHR3.0 or 473 nm for ChR2(E123A) with a multimode fiber coupler for an FC/PC connection). Based on previous work, light output was adjusted to allow for 10 mW at the fiber tip (Deisseroth, 2012; Gradinaru et al., 2009; Huff et al., 2013; Wahlstrom et al., 2020; Wahlstrom et al., 2018; Yizhar et al., 2011), as measured by an optical power meter. Illumination was controlled by a Master-8 stimulator for the ChR2(E123A) (stimulation) experiments or provided continuously for the eNpHR3.0 (inhibition) experiments. The illumination was provided in a separate holding chamber (30 cm x 30 cm x 30 cm) that contained a weighted arm attached to the outside of the chamber with the optical commutator at one end. In all cases, the comparison control was a CaMKIIα-eYFP group that received illumination alone. All illumination was administered bilaterally.

2.4. Behavioral training

2.4.1. Barnes maze

A Barnes maze was used to investigate the consolidation of cued-response and spatial learning. The Barnes maze consisted of an exposed and elevated, brightly lit circular platform (116.8 cm in diameter) with 18 evenly spaced holes (10 cm in diameter) along the perimeter, one of which led to an escape port (Fig. 2C, 3G, and 5C). The platform was covered in black matte plexiglass to provide optimal contrast to the white fur of the rats for automated analysis with NOLDUS Ethovision software. Extra-maze cues consisted of specific symbols on the walls around the maze as well as the general layout of equipment in the room. NOLDUS Ethovision recording software was used to record the time to find the escape port (latency) and the time spent in each quadrant of the maze (duration).

Fig. 2.

Fig. 2.

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The BLA in cued-response memory consolidation (Experiment 1). A, Schematic diagram of BLA injection site (left), incubation time, and optic probe placement in BLA (right). B, Left, Fluorescent image of eYFP expression in the BLA. Scale bar = 500 μm. Right, Cresyl violet staining illustrating damage from the virus injector terminating within the BLA and fiber optic probe implant terminating dorsal to the BLA. Scale bar = 1 mm. C, Illustration of the Barnes maze. For cued-response training, a distinct intra-maze cue was attached directly to the escape port. The escape port and cue were randomly shifted to a different cardinal direction for each training trial. D, Experimental timeline for cued-response training and posttraining optical manipulation of BLA cell bodies. E, Left, Latencies to find the escape port during training for those rats that received BLA cell body inhibition after training. Right, Duration in target quadrant during training trials for the same rats. There were no significant group differences in latencies or duration during training. F, Two days after training, rats were tested for retention in a single 180 s trial. Left, Latencies to locate the escape port during the retention test. Rats that received posttraining inhibition of BLA cell bodies had significantly longer latencies to find the escape port than their eYFP-control counterparts. Right, Duration spent in the target quadrant during the retention test. Rats that had received posttraining inhibition of BLA cell bodies spent significantly less time in the target quadrant than eYFP-controls. G, Latencies (left) and duration in target quadrant (right) of rats that received BLA cell body inhibition 3 h after training. There were no significant differences in either case. *, p < 0.05 compared to eYFP-control values.

Fig. 3.

Fig. 3.

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BLA-NAshell pathway inhibition in cued-response and spatial memory consolidation (Experiment 2). A, Schematic diagram of BLA injection site (left), incubation time, and optic probe placement in NAshell (right). B, Left, Fluorescent image of eYFP expression in the BLA. Scale bar = 500 μm, also applies to middle panel. Middle, Fluorescent image of BLA axons in the NAshell in an eYFP-transduced rat, and fiber optic probe aimed at the NAshell. Right, Cresyl violet staining illustrating damage from the fiber optic probe implant terminating dorsal to the NAshell. Scale bar = 1 mm. C, Experimental timeline for cued-response training and posttraining optical manipulation of the BLA-NAshell pathway. D, Left, Latencies to find the escape port during training for rats that received inhibition of BLA-NAshell pathway after training. Right, Duration in target quadrant during training trials for the same rats. There were no significant group differences in either latency or duration during training. E, Two days after training, rats were tested for retention in a single 180 s trial. Left, Latencies to locate the escape port during the retention test. Rats that received posttraining inhibition of the BLA-NAshell pathway had significantly longer latencies to find the escape port than their eYFP-control counterparts. Right, Duration spent in the target quadrant during the retention test. Rats that received posttraining inhibition of the BLA-NAshell pathway spent significantly less time in the target quadrant compared to eYFP-controls. F, Latencies (left) and duration in target quadrant (right) of rats that were given BLA-NAshell pathway inhibition 3 h after training. There were no significant differences in either case. G, Illustration of the spatial Barnes maze. For spatial learning, extramaze cues surrounded the perimeter, and the escape port remained in the same location on every trial, enabling rats to use a spatial strategy to find the port. H, Experimental timeline for spatial training and posttraining optical manipulation of the BLA-NAshell pathway. I, Latencies (left) and duration in target quadrant (right) of rats that were given inhibition of the BLA-NAshell pathway following spatial training. There were no significant differences in either case. *, p < 0.05 compared to eYFP-control values.

Fig. 5.

Fig. 5.

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BLA-NAshell pathway stimulation in cued-response and IA memory consolidation (Experiment 4). A, Schematic diagram of BLA injection site (left), incubation time, and optic probe placement in NAshell (right). B, Fluorescent image of ChR2 expression in the BLA. Scale bar = 500 μm. C, Experimental timeline for cued-response training and posttraining optical manipulation of the BLA-NAshell pathway (top). Illustration of the cued-response Barnes maze (bottom). D, Two days after training, rats were tested for retention in a single 180 s trial. Left, Latencies to locate the escape port during the retention test. Rats that received posttraining bursts of 20 Hz stimulation of the BLA-NAshell pathway had shorter latencies to locate the escape port than their eYFP-control counterparts, though these values did not reach significance. Right, Duration spent in the target quadrant during the retention test. Rats that received posttraining bursts of 20 Hz stimulation of the BLA-NAshell pathway spent more time in the target quadrant compared to eYFP-controls, though these values did not reach significance. E, Experimental timeline for IA training and posttraining optical manipulation of the BLA-NAshell pathway (top). Illustration of the IA chamber (bottom). Two days after training, rats were tested for retention in a single trial. F, Latencies of rats that received posttraining bursts of 20 Hz stimulation of the BLA-NAshell pathway G, Latencies of rats that received posttraining bursts of 8 Hz stimulation of the BLA-NAshell pathway. There were no significant differences in either case.

Rats were handled individually for 1 min per day for 3 days prior to the start of training and, additionally, on the last day of handling, were placed in the optical illumination holding chamber for 1 min to familiarize the rats with the environment. For cued-response training, a distinct intra-maze cue was attached directly to the escape port (Fig. 2C and 5C). The escape port and cue were randomly shifted to a different cardinal direction for each training trial. Therefore, the extra-maze spatial cues could not be used to locate the escape port during cued-response training. For spatial training, the escape port of the Barnes maze was maintained in the same location relative to the extra-maze cues on each trial (Fig. 3G). The escape port location was randomly chosen and counterbalanced within each group so that no particular direction was associated with a single group.

For cued-response and spatial training, rats underwent multiple trials on the training day (Day 1). For each trial, the rat was placed in the center of the Barnes maze and allowed to freely explore the entire apparatus for 60 s to find the escape port and enter. If a rat entered the escape port prior to the 60 s mark, it was permitted to remain in the escape port for 30 s. If the rat did not enter the escape port within 60 s, it was placed in the escape port and permitted to remain there for 30 s. After each trial, the rat was removed from the escape port and placed in its home cage for 1–2 mins while the maze was wiped with 20 % EtOH to remove olfactory cues. This process was repeated for 4 consecutive trials for inhibition experiments (eNpHR3.0 experiments; Fig. 2D, 3C and 3H) or for 3 consecutive trials for the stimulation experiment (ChR2 experiment; Fig. 5C). One fewer training trial was given in the stimulation experiment to prevent a ceiling effect.

Retention was tested two days later (Day 3) when rats again were placed on the center of the Barnes maze and allowed to freely explore for 180 s. For the cued-response version of the task, the escape port with cue was placed in the same direction as the third training trial for each rat. For the spatial version of the task, the escape port was oriented in the same direction as it had been during training on Day 1. For both versions of the task, the latency to enter the escape port and the duration spent in the target quadrant were used as the indices of retention.

2.4.2. Inhibitory avoidance

Rats were trained and tested on a single-trial step-through IA task similar to previously described work (LaLumiere, Nguyen, & McGaugh, 2004; LaLumiere, Pizano, and McGaugh, 2004; Lingg et al., 2020). The apparatus was a trough-shaped alley (91 cm long and 20 cm deep) divided into two compartments: a non-shock (safe) compartment made of white plastic and illuminated by a table lamp (31 cm), and a dark, shock compartment made of stainless steel (60 cm). A stainless steel retractable door separated the two compartments (Fig. 4C and 5E). The shock compartment plates were connected to an AC shock generator (Lafayette Instrument Company) controlled by a timer. After each training and test trial, the apparatus was wiped with 20 % EtOH to remove olfactory cues.

Fig. 4.

Fig. 4.

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BLA-NAshell pathway inhibition in IA memory consolidation (Experiment 3). A, Schematic diagram of BLA injection site (left), incubation time, and optic probe placement in NAshell (right). B, Fluorescent image of eYFP expression in the BLA. Scale bar = 500 μm. C, Illustration of the IA chamber. Rats received a single inescapable footshock after crossing into the shock compartment on the training day. D, Experimental timeline for IA training and posttraining optical manipulation of the BLA-NAshell pathway. E, Two days after training, rats were tested for retention in a single trial. Rats that received posttraining inhibition of the BLA-NAshell pathway had significantly shorter latencies to cross into the shock compartment compared to their eYFP-control counterparts. F, There were no significant differences in latencies of rats that received BLA-NAshell inhibition 3 h after training. *, p < 0.05 compared to eYFP-control values.

Rats underwent 3 days of handling, as described above. The day after the last handling session, the rats underwent a 2 min context pre-exposure trial where they were permitted to freely explore the IA apparatus to familiarize them with the context (Day 1) (Fig. 4D and 5E). On Day 2, rats underwent a single training trial. Each rat was placed in the light compartment facing away from the retractable door and shock compartment. After ~ 10 s, the retractable door was opened, and the rat was permitted to freely explore the apparatus. When the rat crossed into the shock compartment the retractable door was raised, blocking the rat from leaving the shock compartment. After 20 s, the rat received a single inescapable footshock (inhibition experiments: intensity of 0.8 mA and duration of 1 s; stimulation experiments: intensity of 0.45 mA and duration of 1 s). The different footshock intensities were used to prevent floor and ceiling effects, respectively (Huff et al., 2013; Lingg et al., 2020). The rat remained in the shock compartment for an additional 20 s after which the rat was removed and immediately connected to the laser for 15 min of optogenetic illumination (Lingg et al., 2020). All rats had training latencies less than 30 s.

Retention was tested 2 days after training. Each rat was placed in the light compartment facing away from the shock compartment. After 10 s, the retractable door was opened, and the rat was allowed to freely explore the apparatus. Latency to crossover into the shock compartment with all four paws was used as the measure of retention with a maximum latency of 600 s (LaLumiere, Pizano, & McGaugh, 2004).

2.5. Experimental design

For all experiments, male and female rats were trained and tested in separate cohorts to reduce potential effects of odor cues in the apparatuses.

2.5.1. Experiment 1 – The BLA in cued-response memory consolidation

Previous work in male rats found that pharmacological manipulations of the BLA modulate the consolidation of cued-response learning (Packard et al., 1994). Experiment 1 followed up on this early work by examining whether optogenetically inhibiting the BLA in male and female rats after cued-response learning in a Barnes maze task alters retention. Rats received continuous (15 min) optical illumination of eNpHR3.0-transduced BLA cell bodies immediately following the final training trial (Fig. 2D). To determine whether the observed effects of inhibition were due to delayed effects on the retention test and to confirm the time-limited nature of memory consolidation, a 3 h delay experiment was conducted in which rats received cued-response training and then, 3 h after the last training trial, received optical inhibition akin to that of the main experiment.

2.5.2. Experiment 2 – BLA-NAshell pathway inhibition in cued-response and spatial memory consolidation

Experiment 2 examined whether inhibiting the BLA-NAshell pathway after cued-response or spatial learning in a Barnes maze task alters retention. Rats received continuous (15 min) optical illumination of eNpHR3.0-transduced BLA fibers in the NAshell immediately after training on either task (Fig. 3C and 3H) as done previously in our laboratory (Wahlstrom et al., 2018). Two rats in the eNpHR3.0 group fell off the maze during the retention test. In these cases, the latency at which the rat fell off the maze was used as the retention latency as the actual latency would have been longer. As in Experiment 1, a 3 h delay experiment was conducted in which rats received BLA-NAshell inhibition 3 h after training on the cued-response task.

2.5.3. Experiment 3 – BLA-NAshell pathway inhibition in IA memory consolidation

Experiment 3 examined whether inhibiting the BLA-NAshell pathway after IA learning alters retention. Rats received continuous (15 min) optical illumination of eNpHR3.0-transduced BLA fibers in the NAshell immediately after training (Fig. 4D). The rats used in this experiment had previously been used in the 3 h delay BLA-NAshell cued-response experiment except for 2 naïve rats. As in the previous experiments, a 3 h delay experiment was conducted in which rats received inhibition 3 h after IA training.

2.5.4. ExpeHment 4 – BLA-NAshell pathway stimulation in cued-response and IA memory consolidation

Experiment 4 examined whether stimulating the BLA-NAshell pathway after cued-response learning in a Barnes maze task or after IA learning alters retention. Rats received 15 min of illumination of ChR2-transduced BLA fibers in the NAshell immediately following training in either task (Fig. 5C and 5E). The stimulation parameters were 2 s trains of 20 Hz (cued-response and IA experiments) or 8 Hz (IA experiment) light pulses (pulse duration = 5 ms), given every 10 s.

2.6. Verification of opsin expression and histology

After completion of testing, rats were overdosed with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with phosphate-buffered saline (PBS) followed by PBS containing 4 % paraformaldehyde. Brains were removed and stored in 4 % para-formaldehyde PBS for a minimum of 48 h before sectioning. The brains were coronally sectioned (65–75 μm) on a vibratome and mounted onto gelatin-subbed slides for Nissl staining and immunohistochemical procedures or for fluorescent microscopy. Viral expression in the BLA cell bodies and BLA axons in the NAshell were confirmed by using immunohistochemistry or by using a fluorescent microscope (Fig. 1A). Slides were coverslipped with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories). Verification of optic probes’ placement in the BLA (Experiment 1, Fig. 1B) and NAshell (Experiments 2–4, Fig. 1C) was performed with a standard Nissl stain preparation (Cresyl violet) and light microscopy according to the Paxinos and Watson atlas (Paxinos & Watson, 2014). Rats with less than 50 % viral expression in the BLA or misplaced optic probes in the BLA or NAshell were excluded from the final analyses. Fig. 1B and 1C include a random selection of optic probe termination points due to the lead author leaving the laboratory when such data was requested, resulting in limited time to create these figures. We have used this random selection approach in the past when such a large number of animals were included in a study. Similarly, additional representative images showing expression of axon terminals and fiber optics in Figs. 4 and 5 were omitted as representative histological images of the same neural pathway are present in Fig. 3.

Fig. 1.

Fig. 1.

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A, Diagram of rat BLA and adjacent structures (Paxinos & Watson, 2014) (2.64 mm posterior to Bregma) showing the range of viral expression for experimental animals. Minimum expression represented in red, maximum expression represented in blue, and overlap represented in purple. B, Diagrams of rat brain sections (Paxinos & Watson, 2014) (BLA: 2.40 mm, 2.52 mm, 2.64 mm posterior to Bregma) showing 20 optic probe termination sites (10 on each side) randomly selected from rats included in the final analysis. C, Diagrams of rat brain sections (Paxinos & Watson, 2014) (NAshell: 2.16 mm, 1.80 mm, 1.44 mm, and 1.08 mm anterior to Bregma) showing 72 optic probe termination sites (36 on each side) randomly selected from rats included in the final analysis.

2.7. Statistical analysis

GraphPad Prism 10 was used for all statistical analyses. For Barnes maze behavioral analysis, latencies and time in the target quadrant (duration) during training were analyzed using two-way repeated measures ANOVAs with the Geisser-Greenhouse correction. Latencies and durations in the target quadrant for all Barnes maze behavioral experiments during the retention test were analyzed using t tests with Welch’s correction. For IA behavioral analysis, training and retention latencies were analyzed using t tests with Welch’s correction. Individual data points are included to best reflect the numerical spread of the data. The Grubbs method was used to identify statistical outliers across all experiments. The α level was set to 0.05. All measures are expressed as mean ± SEM, and each group’s n is indicated in the figure within or below its respective bar.

3. Results

For the sake of brevity, the training data for studies using the Barnes maze are only depicted for the immediate posttraining inhibition experiment in each case and statistical analyses for Barnes maze training data are included in Table 1. In all experiments, male and female rats within each group did not significantly differ from each other during training or testing, thus, figures and analyses describe the aggregated data. Statistical analyses comparing sex within each group during training and testing are included in the supplement.

Table 1.

Two-way repeated measures ANOVAs of combined latencies and duration in the target quadrant during training for Barnes maze experiments.

Experiment 1: BLA cell body inhibition following cued-response training
Barnes Maze
Experiment		 Training Trial Group: eNpHR3.0
vs. eYFP		 Interaction
Immediate inhibition:
Training latencies		 F(2.54, 45.78) = 1.64
p = 0.20		 F(1,18) = 0.29
p = 0.59		 F(3, 54) = 0.48
p = 0.70
Immediate inhibition:
Training duration		 F(2.94, 52.91) = 1.29
p = 0.29		 F(1, 18) = 1.14
p = 0.30		 F(3, 54) = 0.96
p = 0.42
3 h delay:
Training latencies		 F(2.71, 56.99) = 4.56
p = 0.0079		 F(1,21) = 1.09
p = 0.31		 F(3, 63) = 1.95
p = 0.13
3 h delay:
Training duration		 F(2.78, 58.40) = 0.66
p = 0.57		 F(1, 21) = 0.33
p = 0.57		 F(3, 63) = 1.25
p = 0.30
Experiment 2: BLA-NAshell pathway inhibition following cued-response and
spatial training
Barnes Maze
Experiment		 Training Trial Group: eNpHR3.0
vs. eYFP		 Interaction
Immediate inhibition:
Training latencies		 F(1.98, 47.46) = 10.11
p < 0.0002		 F(1, 24) = 0.51
p = 0.48		 F(3, 72) = 0.73
p = 0.54
Immediate inhibition:
Training duration		 F(2.61, 62.64) = 4.21
p = 0.012		 F(1, 24) = 1.25
p = 0.28		 F(3, 72) = 0.08
p = 0.97
3 h delay:
Training latencies		 F(1.99, 51.71) = 5.71
p = 0.0058		 F(1.26) = 1.73
p = 0.20		 F(3,78) = 3.02
p = 0.034
3 h delay:
Training duration		 F(2.82, 96.85) = 1.75
p = 0.16		 F(1, 103) = 0.10
p = 0.76		 F(3, 103) = 1.56
p = 0.20
Spatial:
Training latencies		 F(2.04, 36.67) = 6.49
p = 0.0037		 F(1, 18) = 0.84
p = 0.37		 F(3, 54) = 1.14
p = 0.34
Spatial:
Training duration		 F(2.81, 50.55) = 1.80
p = 0.16		 F(1, 18) = 0.19
p = 0.66		 F(3, 54) = 1.16
p = 0.33
Experiment 4: BLA-NAshell pathway stimulation following cued-response
training
Barnes Maze
Experiment		 Training Trial Group: ChR2 vs.
eYFP		 Interaction
20 Hz stimulation:
Training latencies		 F(1.04, 20.69) = 9.44
p = 0.0055		 F(1, 20) = 0.57
p = 0.46		 F(2, 40) = 1.26
p = 0.30
20 Hz stimulation:
Training duration		 F(1.93, 38.69) = 6.32
p = 0.0046		 F(1, 20) = 2.42
p = 0.14		 F(2, 40) = 0.32
p = 0.73
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3.1. Experiment 1 – The BLA in cued-response memory consolidation

Previous pharmacological work in males alone suggests that the BLA modulates cued-response retention (Packard et al., 1994). Therefore, Experiment 1 examined whether optogenetically inhibiting the BLA in males and females after cued-response learning in the Barnes maze task alters retention. To optogenetically inhibit the BLA, virus was injected bilaterally into the BLA and two weeks later optical fibers were implanted just dorsal to the BLA (Fig. 2A-2B). One week after optical fiber implantation surgery, rats underwent training on a cued-response version of the Barnes maze in which a distinct cue was attached directly to the escape port (Fig. 2C). The timeline of behavioral training, optical illumination, and retention testing is depicted in Fig. 2D.

A total of 19 rats (eYFP = 10; eNpHR3.0 = 9) were included in the final analyses for the immediate inhibition experiment. Three were excluded for misplaced fiber optic probes (eYFP = 1; eNpHR3.0 = 2) and one for being a statistical outlier (eYFP = 1). Rats that underwent cued-response Barnes maze training and subsequently received BLA inhibition did not show differences in their training latencies or duration in the target quadrant compared to the eYFP-control rats (Fig. 2E left and right, respectively; Table 1). At the retention test, rats that received immediate posttraining inhibition had significantly higher latencies and lower duration in the target quadrant compared to eYFP-control rats (Fig. 2F left and right, respectively), as indicated by unpaired t tests (latencies: t(9.26) = 3.05, p = 0.013; duration: t(8.90) = 3.10, p = 0.013). Thus, posttraining BLA inhibition impaired retention for cued-response learning.

For the 3 h delay experiment, rats received optical illumination of either eNpHR3.0- or eYFP control-transduced BLA cell bodies 3 h after cued-response training on Day 1. A total of 23 rats (eYFP = 10; eNpHR3.0 = 13) were included in the final analyses. Two were excluded for misplaced fiber optic probes (eYFP = 2) and one for being a statistical outlier (eYFP = 1). Rats that subsequently received BLA inhibition did not show differences in their training compared to eYFP-control rats (Table 1; data not shown). An unpaired t test revealed no significant differences in latencies (t(19.37) = 0.14, p = 0.89) or in time spent in the target quadrant (t(20.98) = 0.09, p = 0.93) during the retention test (Fig. 2G left and right, respectively).

3.2. Experiment 2 – BLA-NAshell pathway inhibition in cued-response and spatial memory consolidation

Experiment 2 examined a potential pathway mechanism by which the BLA modulates cued-response and spatial retention by inhibiting the BLA-NAshell pathway after each type of learning in a Barnes maze task. To optogenetically inhibit the BLA-NAshell pathway, virus was injected bilaterally into the BLA and four weeks later optical fibers were implanted bilaterally targeting the NAshell (Fig. 3A-3B). One week after optic probe implantation, rats underwent 4 training trials on the Barnes maze task, followed immediately or 3 h later by optical illumination. Retention testing occurred 2 d later (Fig. 3C).

A total of 26 rats (eYFP = 13; eNpHR3.0 = 13) were included in the final analyses for the cued-response immediate inhibition experiment. Twenty-seven were excluded for a misplaced fiber optic probe (eYFP = 13; eNpHR3.0 = 14), one for insufficient viral expression (eYFP = 1), and one for being a statistical outlier (eYFP = 1). Rats that underwent cued-response Barnes maze training and subsequent optical illumination of either eNpHR3.0- or eYFP control-transduced BLA axons in the NAshell immediately afterwards did not show differences in latencies or duration in the target quadrant compared to the eYFP-control rats during training (Fig. 3D left and right, respectively; Table 1). At the retention test, unpaired t tests indicated that rats that received immediate BLA-NAshell pathway inhibition had significantly higher latencies (t(23.61) = 2.26, p = 0.034) and lower duration in the target quadrant (t(22.82) = 2.09, p = 0.048) compared to eYFP-control rats (Fig. 3E left and right, respectively). Thus, BLA-NAshell pathway inhibition given after training impaired the retention of cued-response learning.

For the 3 h delay experiment, rats received optical illumination of either eNpHR3.0 or eYFP control-transduced BLA axons in the NAshell 3 h after cued-response training on Day 1. A total of 28 rats (eYFP = 16; eNpHR3.0 = 12) were included in the final analyses. Seven were excluded for misplaced fiber optic probes (eYFP = 2; eNpHR3.0 = 5), two for insufficient viral expression (eYFP = 1; eNpHR3.0 = 1), nine for failure to complete the test trial (eYFP = 5; eNpHR3.0 = 4), and one for receiving less than 15 mins of optical illumination due to an unexpected issue (eYFP = 1). Rats that subsequently received optical illumination of eNpHR3.0-transduced BLA axons in the NAshell did not show differences in their training compared to the eYFP-control rats (Table 1; data not shown). However, a significant interaction was revealed between training trial and group for the training latencies data. Although a post hoc test did not reveal a significant difference between groups during any training trial, the eYFP-control rats had significantly higher latencies during training trials 1 and 2 compared to training trial 4, whereas there were no significant differences across training trials for the eNpHR3.0 group (see statistical analyses in supplement and Fig. S.1.). A mixed-effect analysis was used to analyze the training duration data since one animal was missing a data point due to an issue with the experimental computer during a training trial. At the retention test, an unpaired t test revealed no significant differences in latencies (t(25.96) = 0.10, p = 0.92) or in time spent in the target quadrant (t(25.55) = 0.22, p = 0.83) compared to eYFP-control rats (Fig. 3F left and right, respectively).

The present results reveal that activity in the BLA-NAshell pathway promotes the consolidation of cued-response learning. Previous work from our laboratory suggests that the BLA-mEC pathway positively and negatively modulates spatial and cued-response memory consolidation, respectively (Wahlstrom et al., 2018), raising the question of whether the BLA-NAshell pathway has a similar, if opposite, role with regard to spatial learning. Therefore, rats were trained on a spatial Barnes maze task and received posttraining inhibition of the BLA-NAshell pathway. Fig. 3G and 3H show an illustration of the spatial version of the Barnes maze and the timeline of behavioral training, optical illumination, and retention testing for the spatial experiment, respectively. A total of 20 rats (eYFP = 10; eNpHR3.0 = 10) were included in the final analyses. Six were excluded for misplaced fiber optic probes (eYFP = 3; eNpHR3.0 = 3), and two for failure to complete the test trial (eYFP = 1; eNpHR3.0 = 1). Rats that subsequently received BLA-NAshell pathway inhibition did not show differences in their training compared to eYFP-control rats (Table 1; data not shown). At the retention test, an unpaired t test revealed no significant differences in latencies (Fig. 3I, left; t(16.53) = 0.22, p = 0.83) or duration in the target quadrant (Fig. 3I, right; t(13.23) = 0.61, p = 0.55). Thus, in contrast to the opposing BLA-mEC pathway roles in spatial vs. cued-response learning, BLA-NAshell pathway activity does not appear to influence spatial memory consolidation.

3.3. Experiment 3 – BLA-NAshell pathway inhibition in IA memory consolidation

Previous pharmacological work suggests the BLA and NAshell interact to modulate the consolidation of IA learning (Kerfoot & Williams, 2018; LaLumiere et al., 2005). Therefore, Experiment 3 examined whether inhibiting the BLA-NAshell pathway after IA learning alters retention. As in Experiment 2, virus was injected bilaterally into the BLA and four weeks later optical fiber probes were implanted targeting the NAshell (Fig. 4A-4B). One week after optical implantation surgery, rats were trained on the IA task where they received a single inescapable footshock when they crossed into the shock compartment (Fig. 4C). Fig. 4D depicts the timeline of behavioral training, optical illumination, and retention testing. In both the immediate and 3 h delay IA inhibition experiments, experimental and control groups did not differ from each other during training (statistics in supplement), suggesting there were no pre-existing differences between groups. A total of 27 rats (eYFP = 16; eNpHR3.0 = 11) were included in the final analyses for the immediate inhibition experiment and these rats had previously been used in the 3 h delay BLA-NAshell cued-response experiment except for 2 ï rats. One additional rat was excluded due to receiving less than 15 min of optogenetic illumination due to an unexpected issue (eNpHR3.0 = 1). Rats that received posttraining BLA-NAshell inhibition immediately after training had significantly lower retention latencies compared to eYFP-control rats (Fig. 4E; t(15.07) = 2.65, p = 0.018). For the 3 h delay experiment, rats received BLA-NAshell inhibition 3 h after IA training on Day 2. A total of 23 rats (eYFP = 12; eNpHR3.0 =11) were included in the final analyses for the 3 h delay experiment. Fourteen were excluded for misplaced fiber optic probes (eYFP = 6; eNpHR3.0 = 8), eighteen for insufficient viral expression (eYFP = 11; eNpHR3.0 = 7), and one for receiving less than 15 mins of optogenetic illumination due to an unexpected issue (eYFP = 1). An unpaired t test revealed no significant differences in retention latencies (Fig. 4F; t(19.40) = 0.02, p = 0.98). Thus, BLA-NAshell pathway inhibition immediately after training impaired the retention of IA learning.

3.4. Experiment 4 – BLA-NAshell pathway stimulation in cued-response and IA memory consolidation

The present results suggest that inhibition of the BLA-NAshell pathway impairs retention of both cued-response and IA learning, raising the question of whether stimulating the BLA-NAshell pathway enhances the consolidation of each type of learning. To optogenetically stimulate the BLA-NAshell pathway, virus was bilaterally injected in the BLA and four weeks later optical fibers were implanted targeting the NAshell (Fig. 5A-5B). In the cued-response and initial IA experiment, rats received trains of 20 Hz pulses of optical illumination of either ChR2- or eYFP control-transduced BLA axons in the NAshell immediately after cued-response (Fig. 5C) or IA (Fig. 5E) training. This stimulation frequency was chosen based on prior work suggesting that 20 Hz stimulation is an important frequency for modulating behaviors that are dependent on the BLA-NAshell pathway (Britt et al., 2012; Millan et al., 2017).

A total of 22 rats (eYFP = 11; ChR2 = 11) were included in the final analyses for the 20 Hz stimulation cued-response experiment. Twenty-three were excluded for misplaced fiber optic probes (eYFP = 8; ChR2 = 15), five for insufficient viral expression (eYFP = 5), five for failure to complete the test trial (eYFP = 2; ChR2 = 3), and one for being a statistical outlier (ChR2 = 1). Rats that subsequently received BLA-NAshell stimulation did not show differences in their training compared to eYFP-control rats (Table 1; data not shown). Although retention latencies and duration in the target quadrant for the ChR2 rats were lower and higher, respectively, than those of eYFP-control rats, the differences did not reach statistical significance, as indicated by unpaired t tests (Fig. 5D left and right, respectively; latencies: t(19.37) = 1.60, p = 0.126; duration: t(19.27) = 1.70, p = 0.105).

In the IA stimulation experiments, experimental and control groups did not differ from each other during training, suggesting there were no pre-existing differences between groups (statistical analyses in supplement). A total of 26 rats (eYFP = 13; ChR2 = 13) were included in the final analyses for the 20 Hz stimulation IA experiment. Seven were excluded for misplaced fiber optic probes (eYFP = 1; ChR2 = 6) and nine for insufficient viral expression (eYFP = 4; ChR2 = 5). At the 20 Hz retention test, an unpaired t test revealed no significant differences in retention latencies for rats that received bursts of 20 Hz stimulation of the BLA-NAshell pathway immediately after IA training compared to eYFP-control rats (Fig. 5F; t(23.98) = 0.49, p = 0.63). Because we did not observe an effect of 20 Hz stimulation on IA retention and prior work from our laboratory revealed that 8 Hz BLA-mEC stimulation modulates spatial memory (Wahlstrom et al., 2018), we repeated this experiment in a separate group of rats and stimulated the BLA-NAshell pathway at 8 Hz. A total of 28 rats (eYFP = 15; ChR2 = 13) were included in the final analyses for the 8 Hz stimulation IA experiment. Thirteen were excluded for misplaced fiber optic probes (eYFP = 1; ChR2 = 12), eight for insufficient viral expression (eYFP = 3; ChR2 = 5), one that developed a health concern prior to training (ChR2 = 1), and two for being statistical outliers (eYFP = 1; ChR2 = 1). However, an unpaired t test revealed no significant differences in retention latencies for rats that received bursts of 8 Hz stimulation of the BLA-NAshell pathway immediately after training (Fig. 5G; t(25.45) = 0.10, p = 0.92). Thus, stimulation of the BLA-NAshell pathway at 20 Hz (cued-response and IA experiments) or 8 Hz (IA experiment) did not appear to alter memory consolidation for those kinds of learning.

4. Discussion

The current findings reveal a circuit-based mechanism by which the BLA alters cued-response and IA memory consolidation. An initial experiment in the present study found that optogenetic inhibition of the BLA immediately after cued-response training impaired retention in males and females, confirming earlier work using pharmacological manipulations in males alone (Packard et al., 1994). Furthermore, the present work found that immediate posttraining inhibition of the BLA-NAshell pathway impaired retention for cued-response and IA learning. In contrast, optical inhibition of the same pathway immediately following spatial Barnes maze training had no effect on retention. Surprisingly, BLA-NAshell pathway stimulation had no effect on cued-response or IA retention. Together, these results suggest the ability of the BLA to modulate cued-response and IA memory consolidation is mediated through projections to the NAshell. These findings are, to our knowledge, the first to provide evidence for a role of BLA projections to the NAshell in modulating cued-response and IA learning.

4.1. The BLA-NAshell pathway modulates behaviors of different valences

Previous work from our laboratory found distinct projections from the BLA selectively modulate memory consolidation across multiple memory systems (Huff et al., 2016; Wahlstrom et al., 2018). Although previous anatomical work indicates there is a direct projection from the BLA to the NAshell (Kelley et al., 1982; Kita & Kitai, 1990), the influence of this projection on memory consolidation was unknown. The present work found that the BLA-NAshell pathway modulates IA and cued-response memory consolidation. The BLA-NAshell pathway may be involved in both types of learning because the ventral striatum, and nucleus accumbens in particular, plays an important role in the initial connections between a specific action and an outcome as part of instrumental/goal-directed behavior (Corbit et al., 2001; Dickinson, 1985; Mannella et al., 2013). Furthermore, recent research has debated whether the valence of behaviors differentially involves BLA projections to various downstream brain regions or even distinct BLA-NA projections (Beyeler et al., 2018; Beyeler et al., 2016; Kim et al., 2016; Zhang et al., 2021). Indeed, IA is an aversive learning and memory task whereas the cued-response Barnes maze task may have both rewarding and aversive aspects. That the BLA-NAshell pathway modulates IA and cued-response memory consolidation supports the idea that this projection may be involved in modulating tasks with both aversive and rewarding properties.

4.2. Multiple memory systems

Interestingly, the present work found that BLA-NAshell pathway inhibition did not influence spatial memory consolidation, in which the valence would be similar, if not identical, to the cued-response task, suggesting the existence of other key issues that determine whether this pathway is recruited during memory consolidation. Work on multiple memory systems suggests that the dorsolateral striatum (DLS) selectively influences the consolidation of both cued-response and IA, but not spatial, learning (Kirkby & Kimble, 1968; Neill & Grossman, 1970; Packard & Teather, 1997; Packard et al., 2001; Poldrack & Packard, 2003) whereas the dorsal hippocampus selectively influences the consolidation of spatial, but not cued-response, learning (Packard et al., 1994; Packard et al., 2001). Furthermore, previous research indicates the BLA and DLS modulate cued-response and IA memory consolidation, and this likely occurs through interactions between the two regions (Packard et al., 1994; Packard & Teather, 1998; Packard et al., 2001; Parent & McGaugh, 1994). However, the BLA provides limited inputs to the dorsal striatum (Kelley et al., 1982; Kita & Kitai, 1990), and previous work from our laboratory found no effect of BLA-posterior dorsal striatum pathway stimulation on cued-response learning (Wahlstrom et al., 2018). The present observation that immediate posttraining inhibition of the BLA-NAshell pathway impaired retention for cued-response and IA learning points to this pathway as a potential circuit mechanism by which the BLA influences DLS-based cued-response and IA memory processing.

Indeed, previous research suggests BLA projections to the ventral striatum may influence DLS circuitry and DLS-dependent memories (Ambroggi et al., 2008; Cador et al., 1989; Lipton et al., 2019; Murray et al., 2015). Previous work using pharmacological manipulations found a critical role for interactions between the BLA and the NA, and the NAshell subregion in particular, in modulating IA memory consolidation (Kerfoot & Williams, 2018; LaLumiere et al., 2005; Setlow et al., 2000). Moreover, evidence indicates that electrical NAshell stimulation modulates activity of dopamine neurons that project to the DLS (Wouterlood et al., 2018). Together with the current findings, this points to the idea that the BLA-NAshell pathway may indirectly mediate BLA-DLS interactions during cued-response and IA memory consolidation.

4.3. The BLA-NAshell pathway in spatial memory consolidation

Our prior work found that stimulating and inhibiting BLA projections to the mEC enhances and impairs the consolidation of spatial memories, respectively, but has the opposite effect on cued-response memories (Wahlstrom et al., 2018). Such findings are consistent with the idea that spatial and cued-response memory systems are often in competition with one another (Packard et al., 1994; Packard & Teather, 1997; Poldrack & Packard, 2003) and suggest that distinct BLA projections differentially influence spatial vs. cued-response memory consolidation. Furthermore, early work suggests that spatial learning requires a flexible representation of the environment where animals learn to expect something at a particular location rather than learning to perform a specific action to gain an outcome, as in cued-response learning (Morris, 1981; Tolman et al., 1946a, 1946b). Therefore, the present finding that BLA-NAshell inhibition had no effect on the consolidation of spatial memory is consistent with the idea of multiple memory systems.

4.4. Lack of BLA-NAshell pathway stimulation effects

Although prior work indicates that 20 Hz BLA-NA pathway stimulation modulates a variety of behaviors and is effective at driving neuronal interactions between these two brain regions (Britt et al., 2012; Folkes et al., 2020; Millan et al., 2017; Stuber et al., 2011), the present work found no significant effect of posttraining 20 Hz BLA-NAshell pathway stimulation on cued-response or IA retention. Evidence indicates that memory modulation via optogenetic stimulation likely depends on the specific frequency and that this frequency may differ depending on a variety of circumstances. For example, bursts of 8 Hz, but not 4 or 40 Hz, stimulation of the BLA-mEC pathway enhances spatial memory consolidation (Wahlstrom et al., 2018). Based on this previous work from our laboratory, we examined 8 Hz BLA-NAshell pathway stimulation following IA training. However, such stimulation also had no effect on IA retention in the present work. The lack of stimulation effects raises the possibility that BLA-NAshell pathway stimulation at another frequency, such as 40 Hz, modulates such behaviors. Indeed, prior work indicates the importance of 40 Hz stimulation as well as gamma range oscillations (35–45 Hz) in the consolidation of IA/footshock and stimulus–response learning, respectively (Huff et al., 2016; Huff et al., 2013; Popescu et al., 2009). Nonetheless, considering the previous work effectively using 20 Hz stimulation of BLA inputs to the NAshell, it is not clear why no effects were observed in the present study.

Future research could consider alternative techniques or experimental design to examine the role of BLA-NAshell pathway stimulation on cued-response and IA memory consolidation. One possibility would be to perform electrophysiological recordings of this pathway during memory consolidation and then mimic this firing pattern with optogenetic stimulation. However, it is possible that recapitulating the firing rate of these neurons may also result in null findings since the neurons are presumably already firing at that rate. Rather, using a different technique, such as chemogenetics, may further elucidate these results by enhancing natural patterns of activity during memory consolidation. Furthermore, stimulating the BLA-NAshell pathway during different phases of the experiment, such as during training or the retention test would provide insight into the role of this pathway during acquisition and retrieval of different types of learning. Such future research is necessary to determine the influence of BLA-NAshell pathway stimulation on the consolidation of cued-response and IA learning.

5. Conclusions

The results of the present experiments confirm earlier pharmacological work in males alone (Packard et al., 1994) and indicate the BLA is important in the consolidation of cued-response learning in both males and females. Furthermore, the current work found that immediate posttraining optogenetic inhibition of the BLA-NAshell pathway impaired retention for cued-response and IA, but not spatial, learning, providing evidence that the BLA selectively modulates memories via excitatory projections to the NAshell.

Supplementary Material

1

Acknowledgements

The authors would like to thank Matthew McGregor, Alexa Zimbelman, Aspen Holm, and Hanxiao Liu for their helpful comments on a draft of the manuscript.

Disclosure and Funding

Declarations of interest: none. This work was supported by the National Institutes of Health grants MH118754 (KLW), MH104384 (RTL and CKM), and MH132223 (RTL and JJR).

Footnotes

CRediT authorship contribution statement

Bess Glickman: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Krista L. Wahlstrom: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Jason J. Radley: Resources, Funding acquisition. Ryan T. LaLumiere: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nlm.2024.107988.

Data availability

Data will be made available on request.

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Supplementary Materials

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NIHMS2029352-supplement-1.docx (164.8KB, docx)

Data Availability Statement

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