Orexin receptor-1 in the locus coeruleus plays an important role in cue-dependent fear memory consolidation

Abstract

The noradrenergic (NA) projections arising from the locus ceruleus (LC) to the amygdala and bed nucleus of the stria terminalis have been implicated in the formation of emotional memory. Since NA neurons in the LC (LC-NA neurons) abundantly express orexin receptor-1 (OX1R) and receive prominent innervation by orexin-producing neurons, we hypothesized that an OX1R-mediated pathway is involved in the physiological fear learning process via regulation of LC-NA neurons. To evaluate this hypothesis, we examined the phenotype of Ox1r(-/-) mice in the classic cued and contextual fear-conditioning test. We found that Ox1r(-/-) mice showed impaired freezing responses in both cued and contextual fear-conditioning paradigms. In contrast, Ox2r(-/-) mice showed normal freezing behavior in the cued fear-conditioning test, while they exhibited shorter freezing time in the contextual fear-conditioning test. Double immunolabeling of Fos and tyrosine hydroxylase showed that double-positive LC-NA neurons after test sessions of both cued and contextual stimuli were significantly fewer in Ox1r(-/-) mice. AAV-mediated expression of OX1R in LC-NA neurons in Ox1r(-/-) mice restored the freezing behavior to the auditory cue to a comparable level to that in wild-type mice in the test session. Decreased freezing time during the contextual fear test was not affected by restoring OX1R expression in LC-NA neurons. These observations support the hypothesis that the orexin system modulates the formation and expression of fear memory via OX1R in multiple pathways. Especially, OX1R in LC-NA neurons plays an important role in cue-dependent fear memory formation and/or retrieval.

Figure 1.

Ox1r^−/− mice showed decreased freezing behavior in cued fear-conditioning tests. Wild-type (WT; littermates of Ox1r^−/− mice) (n = 15), Ox1r^−/− mice (n = 16) and Ox2r^−/− mice (n = 15) were subjected to a cued fear-conditioning test. A, Schematic drawing of protocol for cued fear-conditioning test. Timing of CS and US (shown by triangles) presentation is shown. For the cued test period, mice were tested in a new cage different from those used for conditioning, and the same auditory cue (CS) was applied without electric shocks (US). B, Freezing time was counted in every 30 s time window and plotted over time. There was a significant difference between Ox1r^−/− and WT mice during the conditioning session. No statistically significant difference was found between WT and Ox2r^−/− mice. Freezing time during 30 s epochs in the test session was counted before (CS−) and during presentation of the cue (CS+). C, Freezing time counted in every 30 s window, plotted over time. D, Graphic representation of total time during test session itemized for CS− and CS+ periods. Data are shown as mean ± SEM. There was a significant difference during both the presentation of CS and the period of CS−. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2.

Impaired freezing behavior in Ox1r^−/− mice and Ox2r^−/− mice in contextual fear-conditioning test. Contextual fear conditioning was performed to examine the ability of Ox1r^−/− and Ox2r^−/− mice to learn and remember the context in which an electric shock was presented. A, Protocol for contextual fear-conditioning test. For the contextual test, mice were tested in the conditioning cage and stayed there for 300 s. B, C, Freezing time counted in every 30 s epoch, plotted over time. Ox1r^−/− (n = 17) and Ox2r^−/− (n = 15) mice showed significant impairment of freezing time compared with wild-type (WT; n = 19) mice during both the conditioning and test periods. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (Ox1r^−/− mice vs WT), #p < 0.05 and ##p < 0.01 (Ox2r^−/− mice vs WT). D, Graphic representation of total time during test session. **p = 0.01.

Figure 3.

Fos expression in NA neurons in the LC after fear conditioning was decreased in Ox1r^−/− mice. Representative images of dual-labeling immunofluorescent study of Fos and TH are shown in the two left columns (B–F). Immunoreactivity for Fos is shown in red and for TH is shown in green. Quantification of the number of double-positive neurons is shown on the right (B–F). Data are shown as mean ± SEM. A, Schematic representation of experimental design. B, Number of Fos/TH double-positive cells in Ox1r^−/− mice in their home cages is similar to that in wild-type (WT) mice. C, After cued fear conditioning, the number of double-positive cells was significantly lower in Ox1r^−/− mice than in WT mice (n = 5, Ox1r^−/−; n = 6). D, Number of double-positive neurons was significantly lower in Ox1r^−/− mice after cued test (WT; n = 6, Ox1r^−/−; n = 7). E, Number of double-positive neurons was significantly lower in Ox1r^−/− mice after contextual conditioning (WT; n = 4, Ox1r^−/−; n = 4). F, Number of double-positive neurons was significantly lower in Ox1r^−/− mice after contextual test (WT; n = 9, Ox1r^−/−; n = 9). Scale bars, 30 μm. *p < 0.05 and **p < 0.01.

Figure 4.

OX1R in the LC is involved in consolidation of cued fear memory. An AAV with LC-NA neuron-specific promoter carrying ChR2 or OX1R each fused to EYFP was injected bilaterally into the LC in Ox1r^−/− mice to examine the effects of their expression on freezing behavior. We used ChR2 injection for the control group. Then, we performed the cued and contextual fear-conditioning test as shown in Figures 1 and 2, and examined whether LC-NA neuron-specific restoration of OX1R expression in the LC could rescue the fear response. A, Constructs of recombinant AAV vectors carrying OX1R for rescued group or ChR2 for control group. B, A representative image of brain sections of Ox1r^−/− mice prepared from AAV-OX1R-EYFP injection group after behavioral analysis labeled with TH and GFP. Immunoreactivity for TH is shown in red, while that of GFP (OX1R-EYFP) is shown in green. Arrows show examples of colocalization (yellow in merged images). Scale bars: 50 μm (top), 10 μm (bottom). C, Freezing time of Ox1r^−/− mice with ChR2 expression in LC-NA neurons (KO-ChR2, n = 7) and with OX1R expression in LC-NA neurons (KO-OX1R, n = 8) during the cued fear-conditioning period. *p < 0.05, **p < 0.01, ***p < 0.001, wild-type (WT) vs KO-OX1R, #p < 0.05, ##p < 0.01, ###p < 0.001, and WT vs KO-ChR2. D, Effects of OX1R injection on freezing time during test period of cued fear conditioning. E, Graphic representation of results in D. OX1R injection significantly increased freezing behavior of Ox1r^−/− mice (KO-OX1R) specifically in the presence of auditory CS, but did not change it in the absence of CS. We also expressed OX1R in NA-LC neurons in WT mice (WT-OX1R, n = 7). This group did not show any difference compared with WT. F, Freezing time of each group during conditioning session of the contextual fear-conditioning test. There was no significant difference between KO-ChR2 (n = 7) and KO-OX1R (n = 7) groups during the conditioning period. G, Effect of restored expression of OX1R in LC on freezing behavior during the test period of contextual fear conditioning compared with the control group. H, Graphic representation of the results in G. We also expressed OX1R in NA-LC neurons in WT mice (WT-OX1R, n = 7). Data are shown as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5.

Zif 268 expression in the LA after cued fear test was lower in Ox1r^−/− mice. Immunostaining of zif268 in the LA. A, Schematic representation of experimental design. B, A representative image of zif268-positive cells in the LA of wild-type (WT; left) and Ox1r^−/− mice (right). There was no significant difference between genotypes. C, Number of zif268-positive cells after cued fear test was significantly lower in Ox1r^−/− mice compared with WT. **p < 0.01. D, Number of zif268-positive cells in the LA of Ox1r^−/− mice and WT. Scale bar, 30 μm. Data are shown as mean ± SEM.

Figure 6.

NA input from LC through OX1R plays an important role in activation of LA. A, Schematic representation of experimental design. B–E, Immunostaining of zif268 in the LA (C, E) and Fos/TH double-positive cells in the LC (B, D) was examined after cued and contextual fear test in Ox1r^−/− mice with AAV-mediated expression of OX1R (KO-1R) or ChR2 (KO-ChR2) in LC-NA cells. B–E, Representative images showing the LC (B, D) or LA (C, E) of ChR2-injected (left) and OX1R-injected (center) Ox1r^−/− mice. B–E, Right, Shows quantification of positive cells. *p < 0.05. Scale bars: B, D, 50 μm; C, E, 30 μm. Data are shown as mean ± SEM.