Extended Data Fig. 1: Learning-induced reduction of p-eIF2α levels in excitatory and in the SST-expressing sub-population of GABAergic neurons.

a, Schematics of the fear conditioning experiment. b, Illustration of different neuronal subtypes in the CA1 region of the hippocampus. c, Quantitative analyses of immunofluorescence images depicting levels of p-eIF2α in CA1 excitatory neurons 15 min after fear conditioning. In the CA1 region, fear conditioning significantly reduces p-eIF2α in excitatory neurons (t _9.8 = 2.31; n = 6, 6, 12 neurons per 67,600 μm² of mouse CA1). Graph on the right depicts a fraction of excitatory neurons showing a decrease in p-eIF2α (between 25–50% decrease) after fear conditioning. d, The p-eIF2α levels in CA1 PVALB⁺ neurons remain unchanged 15 min after fear conditioning (t _9.17 = 0.112; n = 6, 6, 10-11 neurons per 84,500 μm² of mouse CA1). e, The fear conditioning significantly reduces p-eIF2α in the CA1 SST⁺ neurons (t _9.44 = 2.43; n = 6, 6, 6-7 neurons per 84,500 μm² of mouse CA1). Graph on the right shows that in 39.02% of CA1 SST⁺ neurons, fear conditioning causes 25–50% decrease in p-eIF2α levels. f–h, Representative images of p-eIF2α levels in excitatory (GAD67-negative neurons in CA1 pyramidal layer) and GABAergic neurons (PVALB- and SST-expressing) in naive and fear conditioning mice. Three independent experiments showed similar results. i, Timeline for fluorescent labelling of specific neuronal subtypes in CA1, fear conditioning paradigm and AHA injections to identify newly synthesized proteins. j, To visualize AHA-labelled proteins, the azide group (pink) of AHA is covalently bonded with fluorescent alkyne group (orange) through Cu(I) mediated fluorescent non-canonical amino acid tagging (FUNCAT). k, A 21.67 ± 3.91% increase in protein synthesis was observed in the CA1 excitatory neurons of fear conditioning compared to naive mice using FUNCAT (t _8.14 = 2.85; n = 6, 6, 12 neurons per mouse). Representative images of CA1 excitatory neurons showing FUNCAT in naive and fear conditioning mice. Two independent experiments showed similar results. l, Fear conditioning does not affect general protein synthesis in CA1 PVALB⁺ neurons (t _9.99 = 0.23; n = 6, 6, 6 neurons per mouse). Representative images of GABAergic PVALB⁺ neurons with FUNCAT. Two independent experiments showed similar results. m, In the CA1 SST⁺ neurons, fear conditioning causes 14.33 ± 3.64% increase in AHA labelling and FUNCAT signal intensity (t _9.09 = 2.33; n = 6, 6, 5 neurons per mouse). Representative images of CA1 GABAergic SST-expressing neurons with FUNCAT in naive and fear conditioning mice. Two independent experiments showed similar results. Stratum oriens (S.O.). Data are presented as mean ± s.e.m. in c–e, k–m. p-values by two-tailed unpaired t-test with Welch’s correction in c, e, k and m are indicated. Points represent individual mice. Scale bars: 20 μm.

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