Selective enhancement of fear extinction by inhibiting neuronal adenylyl cyclase 1 (AC1) in aged mice

Abstract

Adenylyl cyclase 1 (AC1) is a selective subtype of ACs, which is selectively expressed in neurons. The activation of AC1 is activity-dependent, and AC1 plays an important role in cortical excitation that contributes to chronic pain and related emotional disorders. Previous studies have reported that human-used NB001 (hNB001, a selective AC1 inhibitor) produced analgesic effects in different animal models of chronic pain. However, the potential effects of hNB001 on learning and memory have been less investigated. In the present study, we found that hNB001 affected neither the induction nor the expression of trace fear, but selectively enhanced the relearning ability during the extinction in aged mice. By contrast, the same application of hNB001 did not affect recent, remote auditory fear memory, or remote fear extinction in either adult or aged mice. Furthermore, a single or consecutive 30-day oral administration of hNB001 did not affect acute nociceptive response, motor function, or anxiety-like behavior in either adult or aged mice. Our results are consistent with previous findings that inhibition of AC1 did not affect general sensory, emotional, and motor functions in adult mice, and provide strong evidence that inhibiting the activity of AC1 may be beneficial for certain forms of learning and memory in aged mice.

Keywords: AC1; Extinction; Remote fear memory; Trace fear memory; hNB001.

Fig. 1

Effects of hNB001 on trace fear memory in aged mice. a Schematic diagram showing trace fear memory performed on aged mice. The CS of a white noise (80 dB, 15 s) was delivered 30 s (trace) before the US of a foot shock (0.75 mA, 0.5 s). Mice were conditioned by 10 CS-trace-US-ITI (210 s) trials for training, and received 10 CS–ITI trials in a novel chamber for test after 24 h of training. The mice were administered hNB001 (30 mg/kg) or saline orally for 7 days before training. hNB001 or saline was taken orally 45 min before test. b No effects of hNB001 on trace fear conditioning in aged mice (Saline, n = 9 mice; hNB001, n = 10 mice, Two-way ANOVA, F _(1,17) = 0.9233, p = 0.3501). c hNB001-treated mice showed no significant difference in freezing during ITI-1 to ITI-7, but significantly reduced freezing during ITI-8 to ITI-10, compared with saline-treated mice during trace fear test (Two-way ANOVA, F _(1,17) = 1.817, p = 0.1953; ITI-8, Student’s t-test, t ₍₁₇₎ = 3.271, p = 0.0045; ITI-9, Student’s t-test, t ₍₁₇₎ = 2.316, p = 0.0333; ITI-10, Student’s t-test, t ₍₁₇₎ = 2.896, p = 0.0100). d Statistical results of trace fear conditioning and test in aged mice with oral hNB001 or saline (Student’s t-test, Training, t ₍₁₇₎ = 0.9609, p = 0.3501; Test, t ₍₁₇₎ = 1.348, p = 0.1953). e hNB001-treated mice showed no significant difference in freezing during the first 7 ITI of trace fear test, compared with saline-treated mice (Student’s t-test, t ₍₁₇₎ = 0.2986, p = 0.7689). f hNB001-treated mice showed significantly reduced freezing during the last 3 ITI of trace fear test, compared with saline-treated mice (Student’s t-test, t ₍₁₇₎ = 3.055, p = 0.0072). *p < 0.05, **p < 0.01

Fig. 2

Effects of hNB001 on recent or remote auditory fear memory in adult or aged mice. a Schematic diagram showing auditory fear memory performed on mice. The CS is a tone (2800 Hz, 85 dB, 30 s). The US is a foot shock (0.75 mA, 2 s) that co-terminated with the tone. Mice were conditioned by three CS/US pairings at 30 s intervals for training. 45 min after oral administration of hNB001 (10 mg/kg) or saline, mice received 3 CS–ITI trials in a novel chamber for recent fear memory test after one day of training (Test 1). The mice were given hNB001 orally twice a day for 30 days. Remote fear memory test was performed again 30 days later (Test 2). b There was no significant difference in the recent fear memory between adult and aged mice, but remote fear memory of aged mice was significantly impaired compared with adult mice (Recent, saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₈₎ = 1.594, p = 0.1283; Remote, saline, n = 7 mice, hNB001, n = 8 mice, Student’s t-test, t ₍₁₃₎ = 4.221, p = 0.0010). c Oral administration of hNB001 for a single or 30 days did not affect recent (left) or remote (right) auditory fear memory in adult mice (Recent, saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₈₎ = 0.2439, p = 0.8101; Remote, saline, n = 7 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₅₎ = 0.9273, p = 0.3685). d Oral administration of hNB001 for a single or 30 days did not affect recent (left) or remote (right) auditory fear memory in aged mice (Recent, saline, n = 9 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₇₎ = 0.6973, p = 0.4950; Remote, saline, n = 8 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₆₎ = 1.296, p = 0.2133). ***p < 0.001

Fig. 3

Effects of continuous administration of hNB001 on remote fear extinction in mice. a Schematic diagram showing fear extinction performed on mice. After auditory fear memory tests, the mice performed fear extinction (12 CS-ITI) once a day for three consecutive days. b hNB001 does not affect remote extinction 1/2/3 in adult mice (Saline, n = 9 mice, hNB001, n = 9 mice, Two-way ANOVA, Extinction 1, F _(1,16) = 1.365, p = 0.2597; Extinction 2, F _(1,16) = 1.792, p = 0.1993; Extinction 3, F _(1,16) = 1.807, p = 0.1976). c hNB001 does not affect remote extinction 1/2/3 in aged mice (Saline, n = 8 mice, hNB001, n = 10 mice, Two-way ANOVA, Extinction 1, F _(1,16) = 0.6108, p = 0.4459; Extinction 2, F _(1,16) = 0.2314, p = 0.6370; Extinction 3, F _(1,16) = 0.6563, p = 0.4298)

Fig. 4

Effects of a single administration of hNB001 on nociception, motor, and anxiety-like behavior in mice. a, b There was no significant difference in hind paw withdrawal to von Frey filaments (a) and response latency of the hot plate test (b) after a single oral administration of 10 mg/kg hNB001 in adult mice, compared with the saline group (Mechanical withdrawal, saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₈₎ = 0.2689, p = 0.7911; Hot plate, saline, n = 8 mice, hNB001, n = 9 mice, Student’s t-test, t ₍₁₅₎ = 1.651, p = 0.1195). c There was no significant difference in motor performance between hNB001- and saline-treatment adult mice (Saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₈₎ = 0.5280, p = 0.6040). d The two squares on the left are representative traces showing the movement of hNB001- and saline-treatment mice in the open field test. The pink box is the central area and the green is the peripheral area. There was no significant difference in motor performance and anxiety-related behavior of the open field test after a single oral administration of hNB001 in adult mice, compared with the saline group (Saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, total distance, t ₍₁₈₎ = 0.6008, p = 0.5555; Time in center, t ₍₁₈₎ = 0.2326, p = 0.8187; Number of center entries, t ₍₁₈₎ = 0.4884, p = 0.6311). e, f There was no significant difference in hind paw withdrawal to von Frey filaments (e) and response latency of the hot plate test (f) after a single oral administration of 10 mg/kg hNB001 in aged mice, compared with the saline group (Mechanical withdrawal, saline, n = 9 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₇₎ = 1.328, p = 0.2017; Hot plate, saline, n = 8 mice, hNB001, n = 8 mice, Student’s t-test, t ₍₁₄₎ = 0.8348, p = 0.4179). g There was no significant difference in motor performance between oral administration of hNB001 and saline in aged mice (Saline, n = 10 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₈₎ = 1.058, p = 0.3039). h The two squares on the left are representative traces showing the movement of hNB001- and saline-treatment mice in the open field test. The pink box is the central area and the green is the peripheral area. There was no significant difference in motor performance and anxiety-related behavior of the open field test after a single oral administration of hNB001 in aged mice, compared with the saline group (Saline, n = 9 mice, hNB001, n = 8 mice, Student’s t-test, total distance, t ₍₁₅₎ = 0.8076, p = 0.4319; Time in center, t ₍₁₅₎ = 1.390, p = 0.1862; Number of center entries, t ₍₁₅₎ = 1.363, p = 0.1959)

Fig. 5

Effects of continuous administration of hNB001 on weight, nociception, motor, and anxiety-like behaviors in mice. a Continuous oral administration of 10 mg/kg hNB001 did not affect the weight of adult mice (Saline, n = 10 mice; hNB001, n = 10 mice, Two-way ANOVA, F _(1,18) = 0.0152, p = 0.9031). b, c Continuous oral administration of hNB001 did not affect hind paw withdrawal to von Frey filaments (b) and response latency of the hot plate test (c) in adult mice (Saline, n = 8 mice, hNB001, n = 10 mice, mechanical withdrawal, Student’s t-test, t ₍₁₆₎ = 0.1582, p = 0.8762; Hot plate, Student’s t-test, t ₍₁₆₎ = 0.7155, p = 0.4846). d Continuous oral administration of hNB001 did not affect motor performance in adult mice (Saline, n = 8 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₆₎ = 1.460, p = 0.1638). e The two squares on the left are representative traces showing the movement of hNB001- and saline-treatment mice in the open field test. The pink box is the central area and the green is the peripheral area. Continuous oral administration of hNB001 did not affect motor performance and anxiety-related behavior of the open field test in adult mice (Saline, n = 8 mice, hNB001, n = 10 mice, Student’s t-test, total distance, t ₍₁₆₎ = 0.6770, p = 0.5081; Time in center, t ₍₁₆₎ = 0.1241, p = 0.9028; Number of center entries, t ₍₁₆₎ = 0.7239, p = 0.4796). f Continuous oral administration of 10 mg/kg hNB001 did not affect the weight of aged mice (Saline, n = 8 mice; hNB001, n = 10 mice, Two-way ANOVA, F _(1,16) = 0.1211, p = 0.7324). g, h Continuous oral administration of hNB001 did not affect hind paw withdrawal to von Frey filaments (g) and response latency of the hot plate test (h) in aged mice (Saline, n = 8 mice, hNB001, n = 10 mice, mechanical withdrawal, Student’s t-test, t ₍₁₆₎ = 0.7895, p = 0.4413; Hot plate, Student’s t-test, t ₍₁₆₎ = 0.4190, p = 0.6808). i Continuous oral administration of hNB001 did not affect motor performance in aged mice (Saline, n = 8 mice, hNB001, n = 10 mice, Student’s t-test, t ₍₁₆₎ = 1.539, p = 0.1434). j The two squares on the left are representative traces showing the movement of hNB001- and saline-treatment mice in the open field test. The pink box is the central area and the green is the peripheral area. Continuous oral administration of hNB001 did not affect motor performance and anxiety-related behavior of the open field test in aged mice (Saline, n = 8 mice, hNB001, n = 10 mice, Student’s t-test, total distance, t ₍₁₆₎ = 0.9894, p = 0.3372; Time in center, t ₍₁₆₎ = 0.2430, p = 0.8113; Number of center entries, t ₍₁₆₎ = 0.1349, p = 0.8944)