Optogenetic Inhibition Reveals Distinct Roles for Basolateral Amygdala Activity at Discrete Time Points during Risky Decision Making

Abstract

Decision making is a multifaceted process, consisting of several distinct phases that likely require different cognitive operations. Previous work showed that the basolateral amygdala (BLA) is a critical substrate for decision making involving risk of punishment; however, it is unclear how the BLA is recruited at different stages of the decision process. To this end, the current study used optogenetics to inhibit the BLA during specific task phases in a model of risky decision making (risky decision-making task) in which rats choose between a small, "safe" reward and a large reward accompanied by varying probabilities of footshock punishment. Male Long-Evans rats received intra-BLA microinjections of viral vectors carrying either halorhodopsin (eNpHR3.0-mCherry) or mCherry alone (control) followed by optic fiber implants and were trained in the risky decision-making task. Laser delivery during the task occurred during intertrial interval, deliberation, or reward outcome phases, the latter of which was further divided into the three possible outcomes (small, safe; large, unpunished; large, punished). Inhibition of the BLA selectively during the deliberation phase decreased choice of the large, risky outcome (decreased risky choice). In contrast, BLA inhibition selectively during delivery of the large, punished outcome increased risky choice. Inhibition had no effect during the other phases, nor did laser delivery affect performance in control rats. Collectively, these data indicate that the BLA can either inhibit or promote choice of risky options, depending on the phase of the decision process in which it is active.SIGNIFICANCE STATEMENT To date, most behavioral neuroscience research on neural mechanisms of decision making has used techniques that preclude assessment of distinct phases of the decision process. Here we show that optogenetic inhibition of the BLA has opposite effects on choice behavior in a rat model of risky decision making, depending on the phase in which inhibition occurs. BLA inhibition during a period of deliberation between small, safe and large, risky outcomes decreased risky choice. In contrast, BLA inhibition during receipt of the large, punished outcome increased risky choice. These findings highlight the importance of temporally targeted approaches to understand neural substrates underlying complex cognitive processes. More importantly, they reveal novel information about dynamic BLA modulation of risky choice.

Keywords: basolateral amygdala; choice; decision making; optogenetics; punishment; risk.

Figure 1.

Design of the RDT. A, Each block consists of 8 forced choice trials and 20 free choice trials. Each free choice trial consists of a deliberation and outcome phase. Rats must nosepoke for the extension of either one lever (forced choice trial) or both levers (free choice trial). A press on one lever yields a small safe reward, and a press on the other yields a large reward accompanied by variable probabilities of footshock punishment. B, Laser delivery occurred at 1 of 5 possible phases during each free choice trial (with laser delivery during each test session taking place in only 1 of the 5 phases). Green bars represent periods of laser delivery.

Figure 2.

Functional validation of eNpHR3.0 in the BLA. A, Two-photon z-series projection of a mCherry-positive BLA neuron filled with biocytin and immunolabeled with Alexa-594. B, Activation of eNpHR3.0 produced a clear outward current in mCherry-positive BLA neurons (n = 11) voltage-clamped at −70 mV. Green bar represents optical stimulation. Shaded area around the average trace represents the SEM. C, Identical activation of eNpHR3.0 in current clamp produced robust hyperpolarization (n = 11). A brief, mild, rebound depolarization was apparent immediately after optical stimulation. This current, likely mediated by HCN channels (Womble and Moises, 1993; Park et al., 2007, 2011; Giesbrecht et al., 2010), had a mean amplitude in current clamp of 4.7 ± 1.3 mV and was almost always insufficient to drive the cells to threshold for action potentials. Green bar represents optical stimulation. Shaded area around the average trace represents the SEM. A small subset of individual sweeps that did have at least one rebound action potential after sustained eNpHR3.0-mediated hyperpolarization were removed from the average traces presented in B, C. D, A representative mCherry-positive BLA neuron that was current-clamped at 0 pA shows an increase in firing rate upon injection of a 150 pA current pulse, which is effectively suppressed during activation of eNpHR3.0.

Figure 3.

eNpHR3.0 expression and optic fiber placement in the BLA. A, Schematic depicting the maximum (light gray) and minimum (dark gray) spread of eNpHR3.0 expression in the BLA. B, Optic fiber placements in the BLA. Black circles represent the tips of the optic fibers. C, Representative micrograph depicting eNpHR3.0 expression and the tip of the optic fiber in the BLA. Dashed white line indicates the borders of the BLA. D, Schematic depicting the maximum (light gray) and minimum (dark gray) spread of mCherry expression in the BLA of control rats. E, Optic fiber placements in the BLA in control rats. Black circles represent the tips of the optic fibers.

Figure 4.

BLA inhibition during deliberation decreases risky choice. A, BLA inhibition decreases choice of the large, risky outcome. B, There were no effects of BLA inhibition on win-stay trials. In contrast, there was a near-significant increase in lose-shift trials upon BLA inhibition. Data are mean ± SEM. *Significant difference. ⁺Trend (p = 0.08) toward a significant difference between inhibition and baseline (no laser) conditions.

Figure 5.

BLA inhibition has no effect on risky choice during other task phases. A, BLA inhibition during delivery of the small, safe outcome did not affect choice of the large, risky outcome. B, BLA inhibition during delivery of the large, unpunished outcome had no effect on choice of the large, risky outcome. C, BLA inhibition during the ITI had no effect on choice of the large, risky outcome. Data are mean ± SEM.

Figure 6.

BLA inhibition during delivery of the large, punished outcome increases risky choice. A, BLA inhibition increased choice of the large, risky outcome. B, There was no effect of BLA inhibition on win-stay performance. In contrast, BLA inhibition decreased lose-shift performance. C, BLA inhibition did not alter the intensity thresholds at which shock elicited a flinch, elevation of 1 or 2 paws, or rapid movement of 3 or all paws. Data are mean ± SEM. *Significant difference between inhibition and baseline (no laser) conditions.

Figure 7.

Laser delivery into the BLA has no effect on risky choice in control rats. A, In rats injected with vectors carrying mCherry alone, laser delivery during deliberation did not affect choice of the large, risky outcome. B, Laser delivery during the large, punished outcome had no effect on choice of the large, risky outcome. Data are mean ± SEM.