A Novel Form of Memory for Auditory Fear Conditioning at a Low-Intensity Unconditioned Stimulus

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short-tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus

Consolidation of auditory fear memories formed by weak unconditioned stimuli requires NMDA receptor activation and de novo

BACKGROUND: Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. We previously showed that the selective ablation of striatal neurons in the adult brain impairs the long-term, but not short-term, memory for auditory fear conditioning with a lower-intensity footshock. This finding raises an intriguing possibility that long-term auditory fear memory may be consolidated in the striatum. RESULTS: There was a significant difference in the freezing responses between two groups of mice subjected to paired and unpaired conditioning, indicating that the auditory fear conditioning with a lower-intensity footshock is an associative learning. Post-conditioning infusion of NMDA receptor inhibitors into the striatum suppressed the consolidation of auditory fear memory when mice were conditioned with a low-intensity footshock. Furthermore, intra-striatum infusion of protein synthesis blocker anisomycin immediately or 1 h after the conditioning prevented the formation of auditory fear memory. On the other hand, the infusion of anisomycin 3 h after conditioning exerted little effect on the auditory fear conditioning, consistent with the presence of a critical time window of protein synthesis for memory consolidation. CONCLUSIONS: These results suggest that NMDA receptors and de novo protein synthesis in the striatum are crucial for the consolidation of auditory fear memory formed with a low-intensity unconditioned stimulus

Striatal dopamine D1 receptor is essential for contextual fear conditioning

Fear memory is critical for animals to trigger behavioural adaptive responses to potentially threatening stimuli, while too much or inappropriate fear may cause psychiatric problems. Numerous studies have shown that the amygdala, hippocampus and medial prefrontal cortex play important roles in Pavlovian fear conditioning. Recently, we showed that striatal neurons are required for the formation of the auditory fear memory when the unconditioned stimulus is weak. Here, we found that selective ablation of striatal neurons strongly diminished contextual fear conditioning irrespective of the intensity of footshock. Furthermore, contextual fear conditioning was strongly reduced in striatum-specific dopamine D1 receptor knockout mice. On the other hand, striatum-specific dopamine D2 receptor knockout mice showed freezing responses comparable to those of control mice. These results suggest that striatal D1 receptor is essential for contextual fear conditioning.ArticleSCIENTIFIC REPORTS.4:3976(2014)journal articl

NeuN-immunohistochemstry.

<p>A, Immunohistochemical analysis for neuronal marker NeuN in control (left) and mutant (right) mice 13 days after mock and RU-486 administration, respectively. Scale bar, 1 mm. B, Higher magnification of NeuN-immunohistochemistry in various brain regions. Scale bars, 0.1 mm. C, NeuN immunoreactive (NeuN⁺)-cell density in the CP after drug administration. <i>n</i> = 8–9 each. D, Densities of NeuN-positive cells in the NAc core (NAcC, open circles) and the NAc shell (NAcS, filled circles) after RU-486 treatment of <i>Gng7^+/mCrePR</i>; <i>+/Eno2-STOP-DTA</i> mice (<i>n</i> = 8–9 each). E, Densities of NeuN-positive cells in the lateral amygdala (LA) of control and mutant mice 22 days after mock and RU-486 treatment, respectively (<i>n</i> = 15 each, <i>F</i>_1,28 = 0.23, <i>P</i> = 0.64, one-way ANOVA). Abbreviations: Au, auditory cortex; CA1, hippocampal CA1 region; CP, caudate putamen; Cx, cortex; GP, globus pallidus; MGN, medial geniculate nucleus of thalamus; NAc, nucleus accumbens; OT, olfactory tubercle; PAG, periaqueductal gray; Sp, septum.</p

Ablation of medium-spiny projection neurons in the striatum of mutant mice.

<p>A, Immunoreactivity for calbindin in the dorsal striatum of control (upper) and mutant (lower) mice. B, Immunoreactivity for tyrosine hydroxylase and substance P in substantia nigra of control and mutant mice. C, Immunoreactivity for GAD and enkephalin in GP of control and mutant mice. Abbreviations: CP, caudate putamen; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars, 1 mm.</p

Impairment of long-term fear memory.

<p>A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses to tone were measured 1 or 3 h and 24 h after conditioning. B, Freezing responses of control (open circles, <i>n</i> = 8) and mutant (filled circles, <i>n</i> = 5) mice 1 h (left) and 24 h (right) after conditioning. C, Freezing responses of control (open circles, <i>n</i> = 6) and mutant (filled circles, <i>n</i> = 4) 3 h (left) and 24 h (right) after conditioning. D, Experimental design. Mice were subjected to auditory fear conditioning with a footshock at 0.3 mA or 0.5 mA. One day after conditioning, the conditioned mice were injected with RU-486 or vehicle. Their freezing responses were measured 14 days after drug treatment. E, Mice were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses of mock-injected (open circles, <i>n</i> = 7) and RU-486-injected (filled circles, <i>n</i> = 8) mice on the conditioning (left) and test (right) days. F, Mice were subjected to auditory fear conditioning with the standard footshock at 0.5 mA. Freezing responses of mock-injected (open circles, <i>n</i> = 6) and RU-486-injected (filled circles, <i>n</i> = 7) mice on the conditioning (left) and test (right) days.</p

Impaired freezing responses of mutant mice after auditory fear conditioning with a low-intensity footshock.

<p>A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning. B, Freezing responses of control (open circles, <i>n</i> = 9) and mutant (filled circles, <i>n</i> = 8) mice on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with the standard intensity of footshock (0.5 mA, an arrow). Solid lines represent tone. C, Freezing responses of control (open circles, <i>n</i> = 8) and mutant (filled circles, <i>n</i> = 11) mice and RU-486-treated Gγ7-mCrePR mice (RU-486 control) (shaded triangles, <i>n</i> = 7) on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with a low intensity of footshock (0.3 mA, an arrow). Solid lines represent tone. D, Current thresholds of control (open bar), RU-486-control (shaded bar) and mutant (filled bar) mice for flinch and jump reactions (<i>n</i> = 6 each).</p

Inducible ablation of striatal neurons.

<p>A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus.</p

Performance of mutant mice in motor tests.

<p>A, Foot print of control (left) and mutant (right) mice. Scale bar, 2 cm. B, Tail suspension test of control (left) and mutant (right) mice. C, Performance of control (open circles, <i>n</i> = 9) and mutant (filled circles, <i>n</i> = 8) mice in the stationary thin rod test. D, Performance of control (open circles) and mutant (filled circles) mice in the accelerating rotarod (<i>n</i> = 8 each). E, Locomotor activity of control (open circles, <i>n</i> = 10) and mutant (filled circles, <i>n</i> = 7) mice in the openfield test.</p