Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons

Abstract

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α₁- and β-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

Keywords: basolateral amygdala; chemogenetic tool; conditioned taste aversion; ionotropic receptor; locus coeruleus; memory retrieval.

Figure 1.

Experimental strategy and transgene expression. A, Strategy for IRNA. The Tg mice expressing IR84a/IR8a genes in specific cell types are treated with exogenous ligands in the brain regions, resulting in the activation of target neurons. B, Structure of the gene cassette encoding GFP-IR84a-2A-IR8a with a signal peptide (SP) downstream of the TH gene promoter. B, BamHI, E, EcoRI, H, HindIII, S, SalI; pA, polyadenylation signal. C, Transgene expression in the LC revealed by GFP immunohistochemistry. 4V, Fourth ventricle. D, Confocal microscopic images of LC sections obtained from double immunohistochemistry for TH/GFP and NET/IR8a. Scale bars: C, 500 μm; D, 20 μm.

Figure 2.

Ligand-induced LC activation in the Tg mice. A, Schematic illustration of strategy for a whole-cell current-clamp recording of a brain slice preparation. Inset shows a typical waveform of NE neurons showing a wide action potential with large afterhyperpolarization. B, Excitatory effect of 0.1% (7.3 mm) PhAc on the membrane potential of a NE neuron obtained from a Tg mouse. C, Depolarization block after excitatory response to PhAc in another neuron in a Tg mouse. D, Lack of effect of PhAc (0.2%) on the membrane potential of a neuron from a non-Tg mouse. E, Bar graphs showing the firing frequency before (pre) and after (post) PhAc application and amplitude of PhAc-induced depolarization of NE neurons in the Tg (n = 12 for firing frequency and n = 17 for depolarization amplitude) and the non-Tg (n = 4) mice. *p < 0.05 vs pretreatment in the Tg mice (paired two-tailed t test), ***p < 0.001 vs the non-Tg mice (unpaired two-tailed t test). F, Expression of IR84a/IR8a complex in HEK293 T cells transduced by a lentiviral vector. Immunohistochemistry for GFP and HA tag detected expression of the two receptor subunits. Scale bar, 50 μm. G, Bar graph showing the current amplitude in GFP⁺ (n = 14) and GFP^– (n = 11) cells. Inset shows mean current traces of the PhAc-induced inward current waveform. Solid and dotted lines indicate the waveform of GFP⁺ and GFP^– cells, respectively. **p < 0.01 versus GFP^– cells. H, Ligand dose responses of the GFP⁺ (n = 12) and GFP^– (n = 8) cells. The amplitudes of the ligand-induced currents were normalized in each cell to the amplitude at 0.01% PhAc. **p < 0.01, ***p < 0.001 versus GFP^– cells at the corresponding ligand concentrations. Data are presented as the mean ± SEM. Individual data points are overlaid.

Figure 3.

In vivo LC activation and increased NE release in the Tg mice. A, Schematic diagram for the strategy of single-unit recording for in vivo electrophysiology. PhAc solution (1%) was pneumatically injected through a glass capillary attached to the recording electrode. B, Firing pattern of an identified NE neuron in the Tg (top) and non-Tg (bottom) mice. The timing of the PhAc injection is indicated. C, Firing rate in the pre- and post-PhAc injection period for the Tg (n = 6) and non-Tg (n = 5) mice. ***p < 0.001 (paired two-tailed t test). D, Firing rate of non-NE neurons in the Tg mice with low and high frequency of the activity in the pre- and post-PhAc injection period (n = 5 or 7 for each type). E, Diagram for the microdialysis for measuring NE release in the ACC in response to LC activation. PhAc solution or PBS was injected into the LC, and dialysis samples were collected from the ACC. F, G, Changes in the extracellular NE level after injection for PhAc (0.2 μl/site) of 0.1% in the Tg mice (F) and 0.4%/0.6% in the Tg and non-Tg mice (G). NE levels are expressed as a percentage of the average baseline levels of each mouse. n = 3 for the 0.1% PhAc experiment, and n = 5 or 6 for each group of the 0.4% and 0.6% PhAc experiments. *p < 0.05, ***p < 0.001 versus PBS; ^†p < 0.05, ^††p < 0.01 versus 0.4% PhAc (Holm–Bonferroni test). H, Diagram for the microdialysis for monitoring NE release in the BLA in response to LC stimulation. PhAc solution or PBS was injected into the LC, and dialysis samples were collected from the BLA. I, Changes in the extracellular NE level after 0.4%/0.6% PhAc injection (0.2 μl/site) in the two kinds of mice. NE levels are expressed as a percentage of the average baseline levels of each mouse. n = 5 or 6 for each group. *p < 0.05, **p < 0.01 versus PBS (Holm–Bonferroni test). Data are presented as the mean ± SEM. J, Placement sites of the recording electrodes (NE and non-NE neurons with low and high frequency) for in vivo electrophysiology. K, L, Placement sites of the injection needles into the LC and dialysis probes into the ACC after treatment with PBS and PhAc of 0.1% (K) or 0.4%/0.6% (L). M, Placement sites of the injection needles into the LC and dialysis probes into the BLA after treatment with PBS and PhAc (0.4%/0.6%). The AP coordinates (in mm) are shown. Scale bar, 1 mm.

Figure 4.

Morphologic analysis of LC neurons after PhAc stimulation. A, Histology of LC NE neurons in the Tg mice after PhAc treatment. Sections through the LC were prepared from the Tg mice used for microdialysis analysis 7 d after unilateral treatment with PBS or PhAc (0.4/0.6%) and stained for TH and NET immunohistochemistry. B, Cell counts for TH- and NET-immunopositive cells. The ratio of the number of cells stained for TH or NET in the treated side relative to the intact side was calculated. Data are presented as the mean ± SEM. Individual data points are overlaid. C, Staining of LC sections with cresyl violet, showing no cell damage against LC neurons in the PhAc-treated side. D, Staining for cell death markers. IBO (1 mg/ml) or PhAc (0.6%) was unilaterally injected into the LC (0.2 μl/site) of the Tg mice. LC sections were stained with TUNEL or immunohistochemistry for activated caspase-3. The LC areas in the top images were fourfold magnified in the lower images. Arrowheads indicate the injection sites into the LC. 4V, Fourth ventricle. Scale bars: A, C, D, 1 mm.

Figure 5.

Ligand-induced LC activation enhances memory retrieval of conditioned taste aversion. A, Schedule of the taste reactivity test. In the conditioning phase, mice were presented 0.5 M sucrose as the CS, followed by an intraperitoneal injection with 0.15 m LiCl as the US (days 1 and 2). Then, the mice were habituated to intraoral (i.o.) infusion with tap water in the test chamber (days 3–5). During the test phase, mice received a bilateral LC injection (0.2 μl/site) of PBS or solution containing PhAc (0.4/0.6%) followed by CS presentation to evaluate the rejection response (day 6). B, Experimental apparatus used for the taste reactivity test. A mouse was placed in the test chamber and infused intraorally through a syringe pump, and the behavior of the animal was monitored from the bottom through an inside mirror using a digital video camera. C, Taste reactivity test showing shorter latency of rejection response by ligand-induced LC activation in the Tg mice. n = 8 or 9 for each group in the Tg mice. n = 8–11 for each group in the non-Tg mice. *p < 0.05, **p < 0.01 versus PBS in the Tg mice (Tukey's HSD test). D, Time course of aversive response number. Rejection responses during the 10 min test period were counted, and the number of responses at a 10 s bin was divided by the number of mice used in each group. Insets show the response number during the early phase (<100 s). E, Total number of aversive responses during the 10 min test. Data are presented as the mean ± SEM. Individual data points are overlaid. F, Placement sites of the injection needles into the LC (PBS and 0.4%/0.6% PhAc) for the taste reactivity test. The AP coordinates (in mm) are shown. Scale bar, 1 mm.

Figure 6.

LC activation does not influence taste sensitivity, general arousal state, and locomotion. A, Taste sensitivity test presenting normal intake of 0.5 m sucrose in unconditioned mice. PBS or PhAc solution (0.6%) was bilaterally injected into the LC (0.2 µl/site), and the fluid intake of 0.5 m sucrose was measured. n = 4–7 for each group. B, Taste reactivity test showing normal hedonic responses to 0.5 m sucrose in unconditioned Tg mice. PBS or PhAc solution (0.6%) was bilaterally injected into the LC, and the latency for hedonic responses was measured. n = 6 for each group. C, Taste reactivity test displaying unaltered aversive responses to 0.2 mm quinine in unconditioned Tg mice. After intra-LC injection of PBS or PhAc solution (0.6%), the latency for rejection responses was measured. n = 8 for each group. D, Locomotor activity. The number of beam breaks was counted for every 10 min block. The total number of beam breaks during a 60 min test period was calculated as locomotor activity during the pretreatment (Pre; blocks −6 to −1) and after PBS treatment (blocks 1–6) and the following 0.6% PhAc treatment (blocks 7–12). n = 7 for each group. Data are presented as the mean ± SEM. Individual data points are overlaid. E–H, Placement sites of the injection needles into the LC (PBS and 0.6% PhAc) for the taste sensitivity test (E), taste reactivity test for hedonic responses (F), taste reactivity test for aversive responses (G), and locomotion test (H). The AP coordinates (in mm) are shown. Scale bar, 1 mm.

Figure 7.

Pharmacological blockade of enhanced memory retrieval by LC activation. A, Taste reactivity test in wild-type mice that received an intra-BLA infusion of PRAZ (0.1 or 0.5 μg/site) and PROP (0.4 or 2.0 μg/site). n = 7–10 for each mouse group. *p < 0.05 versus PBS infusion (Tukey's HSD test). B, Taste reactivity test showing the blockade of shortened response latency in the LC-activated Tg mice by the intra-BLA infusion of PRAZ and PROP at the lower doses (0.1 and 0.4 μg/site, respectively). n = 8 or 9 for each group in the Tg mice. n = 7 for each non-Tg group. *p < 0.05, **p < 0.01 versus PBS in the Tg mice (Tukey's HSD test). C, Locomotion test of the Tg mice that received the microinjection of PhAc (0.6%) into the LC and infusion of PRAZ and PROP (0.1 and 0.4 μg/site, respectively) into the BLA. After the habituation to the open field, the mice received the drug treatments, and then the total number of beam breaks (locomotor activity) during a 60 min period was monitored. n = 4 for each group in the Tg mice. Data are presented as the mean ± SEM. Individual data points are overlaid. D, Placement sites of the injection needles into the BLA (PBS, PRAZ of 0.1 and 0.5 μg/site, and PROP of 0.4 and 2.0 μg/site) for the taste reactivity test. E, F, Placement sites of the injection needles into the LC (0.6% PhAc) and BLA (PBS, 0.1 μg/site PRAZ, and 0.4 μg/site PROP) for the taste reactivity test (E) and locomotion test (F). The AP coordinates (in mm) are indicated. Scale bar, 1 mm.

Figure 8.

Pharmacological inhibition of LC neurons impairs memory retrieval. A, Taste reactivity test showing the lengthened latency of rejection response in wild-type mice by injection of an α₂-adrenergic receptor agonist CLO (10 and 25 ng/site) into the LC. n = 7–8 for each mouse group. **p < 0.01, ***p < 0.001 versus the PBS-treated mice (Tukey's HSD test). B, Locomotion test of the wild-type mice injected with CLO (10 and 25 ng/site) in the LC. Total number of beam breaks (locomotor activity) during a 60 min period after the habituation was measured. n = 6 for each group. C, Restoration of delayed rejection response in the CLO (10 ng/site)-injected mice by intra-BLA infusion of adrenergic receptor agonists MET (0.5 μg/site) and ISO (1.25 μg/site). n = 8 for the MET- and ISO-treated groups; n = 7 for the PBS-treated group. ***p < 0.001 versus the PBS-treated mice (Tukey's HSD test). Data are presented as the mean ± SEM. Individual data points are overlaid. D, E, Placement sites of the injection needles into the LC (PBS and CLO of 10 and 25 ng/site) for the taste reactivity test (D) and locomotion test (E). F, Placement sites of the injection needles in the LC (10 ng/site CLO) and BLA (PBS, 0.5 μg/site MET, and 1.25 μg/site ISO) for the taste reactivity test. The AP coordinates (in mm) are presented. Scale bar, 1 mm.