Fig 2: Behavior of class IIa HDAC-deficient mice. Animals of indicated genotypes were examined at p60. See also Supplementary Fig. 7 and Methods. a Analysis of anxiety-like behavior in the dark–light transfer test. Schematics of experimental setup and averaged transitions to light are shown. Control: n = 19 mice; 4KO: n = 13; 5KO: n = 10; DKO: n = 19. b–d Locomotor activity in the open field setup. b Typical tracks during 30 min sessions. c Averaged times spent in the center zone. d Total travel distances. Control: n = 20; 4KO: n = 15; 5KO: n = 7; DKO: n = 8. e–g Assessment of spatial memory in the Barnes maze. e Schematics of the maze and averaged latencies to escape through the tunnel in each of the four sequential daily training sessions. Control: n = 30; 4KO: n = 13; 5KO: n = 10; DKO: n = 30. f Total travel distances in the maze during training. g Memory retrieval in the probe test with escape tunnel removed. Graph shows averaged percentages of time spent in the correct (target) and other quadrants of the maze. Control: n = 30; 4KO: n = 12; 5KO: n = 11; DKO: n = 29. h Acquisition and extinction of associative fear memory. Mice were allowed to habituate (H) and then subjected to CFC in Context A. Freezing was measured on a daily basis in the same or irrelevant context (Context B). Control: n = 15; 4KO: n = 14; 5KO: n = 11; DKO: n = 11. i Conditional place preference (CPP) test. Graph shows averaged times spent on cocaine-paired floor. Control: n = 27; 4KO: n = 13; 5KO: n = 6§; DKO: n = 29. All quantifications are represented as Mean ± SEM. *p < 0.05 (defined by t-test and ANOVA). Actual p-values are indicated in each graph (vertical for t-test and horizontal for ANOVA). § No sex-related differences in behavior were observed with exception of HDAC5-deficient mice in the CPP test, where only females had preference for cocaine-paired floor

Fig 3: Characterization of HDAC4/5-deficient mice. a Photographs of p60 control, conditional Hdac4 knockout (KO, Hdac4flox/flox/Emx1IRES-Cre), constitutive Hdac5 KO (Hdac5-/-), and Hdac4/5 double knockout (DKO, Hdac4flox/flox/Hdac5-/-/Emx1IRES-Cre) mice subjected to tail suspension test. b Averaged leg clasping indexes. n = 20 mice/genotype. c Gross brain anatomies of p60 control and DKO mice. Coronal brain sections were stained with DAPI and the antibody to a pan-neuronal marker, NeuN. See also Supplementary Fig. 5. d, e, Sections were labeled with antibodies against layer/subtype-specific markers of excitatory neurons, Cux1, Ctip2, and Prox1. Images of the primary somatosensory cortex (d) and the hippocampus (e) are shown. Higher-magnification frames are displayed in inserts. f, g RNA-seq analysis of gene expression in the hippocampus at p60. Volcano plot (f) and the STRING network of differentially expressed transcripts (g) are shown (n = 3 mice/genotype). The following clusters are marked: (1) neurotransmitter receptors and calcium channels; (2) cytoskeleton and scaffolds; (3) protein turnover; (4) membrane trafficking; (5) calcium signaling; (6) G proteins and G protein-coupled receptors; (7) MAP kinases; (8) adenylate cyclases; (9) dopamine and hormone receptors; (10) phosphodiesterases. See also Supplementary Fig. 6 and Data Files 2 and 3. h–k Expression of ERGs in the hippocampus during associative learning. h Analysis of short-term memory performance, assessed by measuring freezing in the same context 1.5 h post CFC. Control/Control, n = 11; Control/CFC, n = 27; DKO/Control, n = 11; DKO/CFC, n = 5. i Images of CA3sp of fear-conditioned control and DKO mice (1.5 h post CFC). Sections were labeled with DAPI and antibodies against Fos and Egr1. j Quantifications of Fos/Egr1-positive cells in CA3sp. n = 3 mice/genotype. k Transcripts were isolated from hippocampi at indicated time points after CFC and expression levels of various ERGs were measured by qPCR. Values from four to five mice per genotype/time point are normalized to 30 min post CFC to highlight the prolonged window of ERG expression in DKOs. *p < 0.05 (t-test). The color coding is the same as in j. All quantifications are represented as Mean ± SEM (error bars). *p < 0.05 (defined by ANOVA and/or t-test)

Fig 4: Acute chemical-genetic control of nuclear repression with destabilized HDAC4. a Schematics of stabilization of DD-nHDAC4 protein with TMP and Rosa26 allele for Cre-dependent expression of DD-nHDAC4 form the CAG promoter (R26floxStop-DD-nHDAC4). See also Supplementary Fig. 8a. b–k DD-nHDAC4 was introduced into excitatory forebrain neurons by crossing R26floxStop-DD-nHDAC4 mice with Camk2aCre. TMP-lactate (300 µg/gm body weight) or control vehicle solutions were administered via intraperitoneal injections. b Brains were isolated at p60 3 h after drug delivery. Cortical and cerebellar protein extracts were probed by immunoblotting with antibodies against FLAG or HDAC4. c, d Time course of DD-nHDAC4 stabilization in the cortex. Immunoblot of samples isolated at different intervals after TMP injection (c) and quantifications of DD-nHDAC4 levels (d, r.u.) are shown. n = 3-4 mice/time point. e Images of DD-nHDAC4 immunofluorescence in CA3sp of vehicle and TMP-treated R26floxStop-DD-nHDAC4/Camk2aCre mice (3 h post injection). See also Supplementary Fig. 8b. f–h Stabilized DD-nHDAC4 represses ERGs. R26floxStop-DD-nHDAC4/Camk2aCre mutants were given single doses of vehicle or TMP. Animals were maintained in home cages (HC) or subjected to CFC (3 h post-injection). After 1.5 h, brains were sectioned and labeled with antibodies against FLAG, Egr1, or Fos. Panels show images of the CA3 (f) and averaged densities of Erg1/Fos-positive cells in this area (g, h). See also Supplementary Fig. 8c. i–k Effects of TMP-inducible nuclear repression on associative memory. p60 R26floxStop-DD-nHDAC4/Camk2aCre and Cre-negative R26floxStop-DD-nHDAC4 mice were treated with vehicle or TMP prior to CFC or prior to contextual memory retrieval, as depicted in i. j Freezing of animals that were given drugs 3 h before learning. Control + vehicle: n = 12; Control + TMP: n = 9; DD-nHDAC4 + vehicle: n = 12; DD-nHDAC4 + TMP: n = 12. k Freezing of animals that were given drugs 3 h before memory recall. Control + vehicle: n = 11; Control + TMP: n = 11; DD-nHDAC4 + vehicle: n = 19; DD-nHDAC4 + TMP: n = 9. All quantifications are represented as Mean ± SEM. *p < 0.05 (t-test and ANOVA). Actual p-values are indicated in each graph (vertical for t-test and horizontal for ANOVA)

Fig 5: Morphologies of dendrites and synapses of HDAC4/5-deficient excitatory neurons. a–e Pyramidal cells in the CA1 of DKO and control Emx1IRES-Cre mice were visualized by Cre-inducible expression of membrane-bound GFP from an adeno-associated virus (AAV DJ DIO-mGFP). Animals were injected with AAVs at p45 and analyzed by confocal imaging of brain sections at p60. a Schematic diagram of AAV recombination. b 3D reconstructions of dendritic trees of individual glutamatergic neurons. c Quantifications of branch orders (NBO), trees, nodes, ends, tree length (TL, µm × 100), and complexity indexes (Com, a.u. × 10,000). Averaged values are from 3 mice/15 neurons per genotype. d Pseudo-colored images of postsynaptic spines on proximal dendrites in CA1sr. e Linear densities, surface areas, and volumes of all and mushroom (M)-type spines. Control: n = 3 mice/17 neurons; DKO: n = 3/21. f, g EM tomography analysis of architectures of glutamatergic synapses in CA1sr. f Top panels: 3D reconstructions of spines, postsynaptic densities (PSDs), nerve terminals, and synaptic vesicles (SVs). Bottom panels: isolated 3D reconstructions of PSDs. g Average volumes and surface areas of PSDs, total SV pools, and numbers of vesicles tethered to active zones (RRP). Control: n = 3 mice/14 synapses; DKO: n = 3/14. All quantifications are represented as Mean ± SEM. *p < 0.05 (defined by t-test). Actual p-values are indicated in each graph