Male Sprague-Dawley rats (2–3 months), serotonin_2A receptor knockout (5-HT_2A^–/–) mice (Weisstaub et al., 2006) and wild-type (WT) littermate controls (4–5 months) maintained on a 129S6/SvEv background were bred in the Tata Institute of Fundamental Research (TIFR) animal facility and CREB deficient/knockout (CREBαδ KO) mice were bred in the University of Pennsylvania animal facility and used for all experiments. For experiments using CREB deficient mice, the hypomorphic CREBαδ knockout mouse line that lacks the α and δ isoforms of CREB (CREBαδ KO) were used (Blendy et al., 1996). CREBαδ KO mice were generated as previously described (Walters and Blendy, 2001) and were maintained as F1 hybrids of 129SvEvTac:C57BL/6. For all experiments, CREBαδ KO mice and the WT controls were generated via crossing heterozygote CREBαδ 129SvEvTac with heterozygote CREBαδ C57BL/6 mice, allowing for a uniform genetic background in the experimental cohort. For the establishment of in vitro cortical cultures, rat embryos were derived at embryonic day 17.5 (E17.5) from timed pregnant Sprague-Dawley dams. All animal procedures using Sprague-Dawley rats and serotonin_2A receptor knockout (5-HT_2A^–/–) mice were carried out in accordance with the Committee for Care and Supervision of Experimental Animals (CPCSEA) and approved by the TIFR Institutional Animal Ethics Committee. All experiments with the CREBαδ KO mouse line were carried out in accordance with the NIH guideline for the care and use of laboratory animals and were approved by the University of Pennsylvania Animal Care and Use Committee. Animals were group housed and maintained on a 12 h light–dark cycle (lights on at 7 am) with access to food and water ad libitum.

Sprague-Dawley rats, CREBαδ KO and litter-matched WT mice, received intraperitoneal injections of the 5-HT_2A agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI, 8 mg/kg, Sigma-Aldrich, United States) or vehicle (0.9% NaCl) (Vaidya et al., 1997). 5-HT_2A^–/– and litter-matched WT mice received intraperitoneal administration of DOI (2 mg/kg) or vehicle (0.9% saline) and animals were sacrificed 2 h post drug administration (Vaidya et al., 1997; Benekareddy et al., 2012).

To make comparisons with a rapid-action antidepressant treatment, we also subjected a cohort of Sprague Dawley rats to electroconvulsive seizure (ECS) treatment via ear-clip electrodes (ECS unit, UGO Basile, Comerio, Italy) (frequency: 100 pulses/s; pulse width: 0.9 ms; pulse duration: 0.5 s; current: 80 mA). ECS animals were subjected to a single ECS treatment while sham-treated control animals underwent the application and removal of ear-clip electrodes without electrical stimulation, and were sacrificed 2 h post treatment. Animals were decapitated immediately, neocortex was dissected and snap frozen in liquid nitrogen.

Primary cortical neuron cultures were established from E17.5 rat embryos as described previously (Desouza et al., 2011; Fanibunda et al., 2019). Rat embryonic cortices were dissected and treated with trypsin-EDTA for 10 min, prior to dissociation in culture medium - Neurobasal medium supplemented with 2% B27 supplement, 0.5 mM L-glutamine, 5 U/ml penicillin and 5 U/ml streptomycin (Invitrogen, United States). Cells were plated on poly-D-lysine (Sigma-Aldrich, United States) coated 35 mm dishes at a density of 10⁶ cells/dish. Following attachment and neurite extension in vitro for a period of 7 days, neurons were treated with DOI (10 μM) or vehicle (DMSO) for 2 h on day in vitro (DIV) 10. In experiments to delineate signaling events downstream of 5-HT_2A receptor activation, neurons were treated with DOI (10 μM) for 2 h in the presence of the 5-HT_2A receptor antagonist, MDL100,907 (10 μM) and specific signaling pathway inhibitors, namely the phospholipase C (PLC) inhibitor U73122 (5 μM), the mitogen activated protein kinase kinase (MAPKK) inhibitor U0126 (50 μM) and the CaM kinase II (CaMKII) inhibitor KN-62 (10 μM) (Tocris Bioscience, United Kingdom). The inhibitors were added to the cultures 30 min prior to DOI treatment, and were present throughout the duration of DOI exposure. Following treatments, cortical neurons were processed for immunofluorescence, RNA extraction for qPCR analysis, or western blot analysis.

Immunofluorescence staining was performed as described previously (Fanibunda et al., 2019). In brief, cortical neurons were fixed in 4% paraformaldehyde, followed by blocking in 10% horse serum and incubated with primary antibodies, rabbit anti-pCREB (1:1000; Cell Signaling Technology, MA, United States) or goat anti-5-HT_2A receptor (1:500, Santa Cruz Biotechnologies, United States) along with the pan-neuronal marker, mouse anti-MAP2 (1:1000, Sigma-Aldrich, United States) overnight at 4°C. This was followed by incubation with secondary antibodies, Alexa 488 conjugated anti-goat (1:500; Molecular probes, CA, United States) or Alexa 488 conjugated anti-rabbit (1:500; Molecular Probes, CA, United States) or biotinylated horse anti-mouse (1:500, Roche Applied Science, Switzerland) with subsequent incubation with streptavidin-conjugated Alexa 568 (1:500, Molecular Probes, CA, United States) for 2 h. Following secondary antibody incubation and serial washes, cortical neurons were mounted in Vectashield (Vector Laboratories, CA, United States), and images were captured on the Zeiss LSM5 Exciter laser scanning microscope.

Rat cortical neurons were lysed in Laemmli sample buffer (2% SDS, 10% glycerol, 60 mM Tris-Cl, 0.01% bromophenol blue) and proteins were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene fluoride (PVDF, GE Healthcare, United Kingdom) membranes. Blots were blocked in 5% fat-free milk in 0.05% Tris Buffered Saline-Tween 20 (TBS-T) and incubated in primary antibodies in 0.05% TBS-T, overnight at 4°C. Primary antibodies included rabbit anti-pCREB (1:500; Cell Signaling Technology, MA, United States) and rabbit anti-CREB (1:1000, Cell Signaling Technology). Blots were washed 3–5 times and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody (GE Healthcare, United Kingdom) for 1 h. Protein-antibody complexes were detected on X-ray films following addition of Enhanced Chemiluminescence (ECL) substrate (GE Healthcare, United Kingdom). The relative density of the pCREB and CREB bands was quantitated using ImageJ software (NIH, United States), and was represented as a pCREB/CREB ratio.

RNA was isolated using Tri Reagent (Sigma-Aldrich, United States), according to the manufacturer’s protocols. Two μg of RNA per sample was reverse transcribed using a complementary DNA (cDNA) synthesis kit (QuantiTect reverse transcription kit, Qiagen, Germany). Quantitative PCR (qPCR) was performed in a Mastercycler^® ep realplex real-time PCR system (Eppendorf, Germany). cDNA was amplified using a SYBR Green kit (Applied Biosystems, CA, United States), with primers for the genes of interest (Supplementary Table 1). Hypoxanthine phosphoribosyl transferase (Hprt) was used as an endogenous housekeeping gene control for normalization. The relative expression levels between control and treated samples were computed by the comparative Ct method, as described previously (Schmittgen and Livak, 2008). Data are represented as fold change ± SEM as compared to control.

Chromatin immunoprecipitation (ChIP) was carried out as described previously (Desouza et al., 2011). Briefly, bilateral frontal neocortices were dissected and fixed to crosslink the DNA with the bound proteins. The tissue was placed in a pre-chilled Dounce homogenizer, sonicated and immunoprecipitated using a phosphorylated CREB (pCREB) antibody (1 μg; Cell Signaling Technology, MA, United States). Following reverse crosslinking and chromatin precipitation, qPCR analysis was performed for upstream sequences of the Arc, BdnfI, Cebpb, cFos, Egr1, and Egr2 promoters that contained putative CRE sites as predicted using AliBaba 2.1¹. For each sample the results were normalized to the input chromatin from the same sample. (Primer sequences used in ChIP experiments: Supplementary Table 2).

Administration of DOI results in a stereotypical head twitch behavior, characterized by rapid radial movements of the head (Canal and Morgan, 2012; Halberstadt and Geyer, 2013). This behavior was videotaped in the home cage for a total duration of 20 min, commencing 20 min following administration of the DOI or vehicle (saline) treatment. The total number of head twitches in this time window were counted by an experimenter blind to the experimental treatment groups.

CREBαδ KO and litter-matched WT mice were administered DOI and vehicle (Veh) resulting in four groups: WT + Veh, WT + DOI, CREBαδ KO + Veh, CREBαδ KO + DOI. Mice were anesthetized with sodium thiopentone and transcardially perfused with 4% paraformaldehyde (PFA). The brains were postfixed in 4% PFA overnight and cryoprotected in 30% sucrose in 4% PFA prior to being shipped to TIFR, India. Coronal sections of 30 μM thickness were cut on the freezing microtome (Leica Biosystems, Germany), fixed, blocked and acetylated. The floating sections were incubated for 20 h at 60°C in a hybridization buffer (50% formamide, 1xSSC, 25xDenhardt’s solution, 40 mM dithiothreitol, 150 μg/ml yeast tRNA, 10% dextran sulfate, 400 μg/ml salmon sperm DNA) containing ³⁵S-UTP labeled antisense riboprobes for Arc mRNA at a concentration of 1 × 10⁶ cpm/300 μl. Antisense riboprobes to Arc mRNA were generated from a transcription-competent plasmid kindly provided by Dr. Oswald Steward (University of California, Irvine, CA, United States). Following hybridization, all sections were washed in ribonuclease A (20 mg/ml; USB corporation, United States), followed by stringent washes in decreasing concentrations of SSC, mounted on slides, air dried and exposed to Hyper film β-max (GE Healthcare, United States) for 7 days. Levels of Arc mRNA were quantified using Scion Image (Scion, United States) and calibrated using ¹⁴C standards to correct for non-linearity. Equivalent areas of the somatosensory and prefrontal cortex were outlined and optical density measurements were determined (3–4 sections/animal).

Results were subjected to statistical analysis using Student’s unpaired t-test for experiments with two groups (GraphPad InStat) and one-way ANOVA (GraphPad, Prism 8) for experiments using signaling pathway inhibitors, followed by Tukey’s post hoc test for group comparisons. For four group experiments statistical analysis was performed using two-way ANOVA (GraphPad, Prism 8). Tukey’s post hoc test for group comparisons was applied only when there was a significant two-way ANOVA interaction observed between the two variables of DOI treatment and 5-HT_2A^–/– KO genotype or DOI treatment and CREBαδ KO genotype. Statistical significance was determined at p < 0.05.

The 5-HT_2A receptor agonist DOI, is a potent hallucinogen that is known to evoke an increase in the expression of several neuronal plasticity-associated genes (González-Maeso et al., 2003). Following ligand binding, the Gq-coupled 5-HT_2A receptor can differentially recruit multiple signaling pathways to bring about distinct signaling responses. We sought to address the contribution of the 5-HT_2A receptor, the Gq-coupled phospholipase C (PLC) signaling pathway, the MAP kinase and CaM Kinase II (CaMKII) signaling pathways and the transcription factor CREB to the DOI-evoked induction of neuronal plasticity-associated gene expression. We stimulated primary rat cortical neurons in vitro with DOI, in the presence or absence of the 5-HT_2A receptor antagonist, MDL100,907; PLC inhibitor, U73122; CaMKII inhibitor, KN-62 or the MAPKK inhibitor, U0126 (Figure 1A). We observed an increase in transcript expression of Arc, Bdnf1, Cebpb, cFos, Egr1, and Egr2 mRNA levels following DOI treatment, which was inhibited by the 5-HT_2A receptor antagonist MDL100,907, as well as the PLC signaling pathway inhibitor, U73122 (Figure 1B) {one-way ANOVA: Arc: [F₍₃,₁₂₎ = 11.23, p = 0.0008]; Bdnf1: [F₍₃,₁₂₎ = 90.59, p < 0.0001]; Cebpb: [F₍₃,₁₂₎ = 14.78, p = 0.0002], cFos: [F₍₃,₁₂₎ = 135.6, p < 0.0001], Egr1: [F₍₃,₁₂₎ = 26.17, p < 0.0001], Egr2: [F₍₃,₁₂₎ = 28.45, p < 0.0001]}. These observations indicate that the 5-HT_2A receptor mediates the DOI-evoked induction in neuronal plasticity-associated genes, via recruiting the Gq-coupled PLC pathway. We next sought to delineate the contribution of the downstream MAP kinase and CaM Kinase II (CaMKII) signaling pathways to the DOI-evoked upregulation of neuronal plasticity-associated genes. We observed an upregulation of Arc, Bdnf1, Cebpb, and Egr2 mRNA levels following DOI treatment, which was inhibited by both the CaMKII inhibitor KN-62 and the MAPKK inhibitor U0126 (Figure 1C) {one-way ANOVA: Arc: [F₍₃,₉₎ = 7.551, p = 0.008]; Bdnf1: [F₍₃,₁₁₎ = 6.436, p = 0.009]; Cebpb: [F₍₃,₈₎ = 5.293, p = 0.027], Egr2: [F₍₃,₁₀₎ = 7.685, p = 0.006]}. In contrast, the DOI-mediated upregulation of cFos mRNA levels was prevented by only the CaMKII inhibitor KN-62, while the increase in the Egr1 transcript levels was abrogated exclusively by the MAPKK inhibitor U0126 (Figure 1C) {one-way ANOVA: cFos: [F₍₃,₁₁₎ = 5.563, p = 0.014], Egr1: [F₍₃,₁₁₎ = 14.02, p = 0.0004]}. This indicates a differential involvement of the MAP kinase and the CaMKII pathway in the 5-HT_2A receptor-mediated transcriptional regulation of specific neuronal plasticity-associated genes (Figure 1D).

We next sought to address the possible role of the transcription factor CREB in mediating the signaling events evoked by DOI, downstream of the 5-HT_2A receptor. The DOI regulated genes, Arc, Bdnf1, Cebpb, cFos, Egr1, and Egr2 were found to contain putative cAMP response element (CRE) (TGACG/CGTCA/TGACGTCA) sites in the upstream promoter regions, suggestive of the possibility of a CREB-dependent transcriptional regulation. We first confirmed 5-HT_2A receptor expression in cortical neurons, by immunofluorescence staining (Figure 1E). Immunostaining with the phosphorylated CREB (pCREB) antibody demonstrated an increase in pCREB immunofluorescence intensity in DOI-treated rat cortical neurons as compared to vehicle-treated control neurons (Figure 1F). Immunoblotting to detect pCREB levels, also demonstrated a robust increase in pCREB/CREB levels in DOI-treated cortical neurons (Figure 1G), further corroborating that DOI treatment enhances pCREB levels. Further, the MAPKK inhibitor (Figure 1H) and the CaMKII inhibitor (Figure 1I), both prevented the DOI-evoked increase in pCREB/CREB levels, demonstrating a role for the MAP kinase pathway and CaMKII pathway in mediating the DOI-evoked increase in pCREB levels (Figure 1J) [pCREB/CREB: F₍₃,₁₇₎ = 5.561, p = 0.008]. This data collectively suggests that DOI, a hallucinogenic ligand of the Gq-coupled 5-HT_2A receptor, recruits the PLC pathway driving MAP kinase and CaMKII signaling to enhance the phosphorylation of the transcription factor CREB in rat cortical neurons (Figure 1K).

Given that the hallucinogen DOI, in addition to exhibiting agonist action at the 5-HT_2A receptor, also binds to the 5-HT_2C receptor with lower affinity (Titeler et al., 1988), we further sought to evaluate the contribution of the 5-HT_2A receptor to the DOI-mediated induction of neuronal plasticity-associated genes in 5-HT_2A^–/– receptor deficient mice (Figure 2A). We confirmed that 5-HT_2A^–/– mice exhibit both a robust reduction in cortical 5-HT_2A, but not in 5-HT_2C, mRNA expression (Figure 2B), accompanied by a significant decline in a stereotypical head twitch response (HTR) behavior evoked by the 5-HT_2A receptor agonist DOI (Canal and Morgan, 2012; Halberstadt and Geyer, 2013). Two-way ANOVA analysis for HTR events (Figure 2C) indicated a significant DOI by 5-HT_2A^–/– genotype interaction [F₍₁,₂₉₎ = 113.4, p < 0.0001], as well as significant main effects of DOI [F₍₁,₂₉₎ = 189.1, p < 0.0001] and 5-HT_2A^–/– genotype [F₍₁,₂₉₎ = 107.6, p < 0.0001].

We next performed qPCR analysis to assess whether the DOI-evoked upregulation of specific neuronal plasticity-associated genes Arc, Bdnf1, Cebpb, cFos, Egr1, and Egr2 mRNA expression observed in vitro, was altered in the neocortex of WT and 5-HT_2A^–/– mice following acute DOI administration (Figures 2D–I). We noted significant two-way ANOVA interactions of DOI by 5-HT_2A^–/– genotype for the neocortical mRNA expression of Arc [F₍₁,₄₄₎ = 4.920, p = 0.0318], Bdnf1 [F₍₁,₄₄₎ = 4.45, p = 0.04], cFos [F₍₁,₄₄₎ = 12.59, p = 0.0009], Egr1 [F₍₁,₄₄₎ = 4.257, p = 0.045] and Egr2 [F₍₁,₄₄₎ = 1118, p < 0.0001], but not for Cebpb mRNA expression [F₍₁,₄₄₎ = 2.49, p = 0.12]. We also noted significant main effects of DOI for Arc [F₍₁,₄₄₎ = 81.38, p < 0.0001], Bdnf1 [F₍₁,₄₄₎ = 20.37, p < 0.0001], Cebpb [F₍₁,₄₄₎ = 8.518, p = 0.0055], cFos [F₍₁,₄₄₎ = 26.72, p < 0.0001], Egr1 [F₍₁,₄₄₎ = 34.58, p < 0.0001], and Egr2 [F₍₁,₄₄₎ = 1392, p < 0.0001] mRNA expression in the neocortex. Significant main effects of 5-HT_2A^–/– genotype were observed for neocortical mRNA levels of Arc [F₍₁,₄₄₎ = 15.79, p = 0.0003], Bdnf1 [F₍₁,₄₄₎ = 15.77, p = 0.0003], Cebpb [F₍₁,₄₄₎ = 7.399, p = 0.009], and Egr2 [F₍₁,₄₄₎ = 1122, p < 0.0001], but not for cFos and Egr1 mRNA expression. Post hoc Tukey comparison analysis revealed that the DOI-evoked upregulation of Bdnf1 and cFos expression noted in the neocortex of WT mice was completely abrogated in 5-HT_2A^–/– receptor deficient mice, and the DOI-evoked increase in Arc, Egr1, and Egr2 neocortical mRNA levels noted in WT mice was significantly attenuated in the 5-HT_2A^–/– receptor deficient genotype. Collectively, studies with pharmacological blockade of the 5-HT_2A receptor in vitro, and using 5-HT_2A^–/– mice in vivo, indicate the critical contribution of the 5-HT_2A receptor to the DOI-mediated regulation of a subset of neuronal plasticity-associated genes in the neocortex.

Given that we observed that the 5-HT_2A receptor agonist, DOI, induced a robust upregulation of neuronal plasticity-associated genes in rat cortical neurons in culture, as well as in WT but not 5-HT_2A^–/– mice, we next examined whether DOI administration to Sprague-Dawley male rats could evoke similar alterations in neuronal plasticity-associated genes in the neocortex. We systemically administered DOI (8 mg/kg) and evaluated gene expression in the neocortex at a 2 h time-point post treatment (Figure 3A). DOI administration evoked a robust increase in the gene expression of Arc, Atf3, Atf4, Bdnf1, Cebpb, Cebpd, Egr1, Egr2, Egr3, Egr4, cFos, JunB, and Nfkbia in the rat neocortex (Figure 3B).

We next examined if a subset of the genes upregulated by DOI treatment, that are known to contain putative CRE binding sites in their promoter regions based on in silico analysis, also exhibit enrichment for pCREB within their promoters (Figure 3C). Enhanced expression of Arc, Bdnf1, Cebpb, and cFos in the neocortex of DOI-treated animals was accompanied by a significant enrichment of pCREB at the promoters of Arc, Bdnf1, Cebpb, and cFos (Figure 3D). In contrast, the enhanced gene expression of Egr1 and Egr2 was not associated with any significant change in pCREB enrichment at putative CRE sites within their promoter regions (Figure 3D). Taken together, these results indicate that in vivo administration of the 5-HT_2A receptor agonist DOI, evokes a robust upregulation of several neuronal plasticity-associated genes in the neocortex, a subset of which exhibit significant pCREB enrichment in their promoter regions.

To draw a comparison of the nature and magnitude of regulation of this subset of neuronal plasticity-associated genes in the neocortex by the serotonergic hallucinogen, DOI, with that evoked by a rapid-action antidepressant treatment, electroconvulsive seizure (ECS) treatment, neocortices derived from acute ECS or sham treated rats were subjected to qPCR analysis (Supplementary Figure 1). We noted a robust increase in Arc, Bdnf1, Cebpb, cFos, and Egr2 but not Egr1 mRNA levels following acute ECS treatment (Supplementary Figure 1), with the nature and scale of upregulation comparable to that following DOI treatment.

Given the evidence both in vitro and in vivo, that the 5-HT_2A receptor agonist, DOI induces (1) a robust increase in pCREB levels in rat cortical neurons, (2) a significant enrichment of pCREB at the promoters of specific plasticity-associated genes that are robustly enhanced by DOI treatment, we next sought to address the contribution of CREB to the regulation of these transcripts. We used hypomorphic CREBαδ KO mice (Blendy et al., 1996) that are reported to have a greater than 90% reduction in CREB binding activity to consensus CRE target sites (Walters and Blendy, 2001). We treated WT and CREBαδ KO mice with vehicle or DOI (8 mg/kg), and assessed transcript expression of specific neuronal plasticity-associated genes at a 2 h time-point post treatment (Figure 4A). To rule out the possibility that CREB may indirectly regulate 5-HT_2A or 5-HT_2C receptors, we first assessed whether the baseline expression of these receptors was altered in CREBαδ KO mice as compared to their WT controls, and observed no change in 5-HT_2A or 5-HT_2C receptor mRNA levels within the neocortex (Figure 4B). Further to assess whether CREB deficient mice exhibit any change in 5-HT_2A receptor evoked behavioral responses, we evaluated the HTR behavior evoked by DOI. Behavioral analysis to quantify the number of HTR events in WT and CREBαδ KO mice, indicated that the number of HTR responses evoked by DOI were unaltered in CREBαδ KO mice (Figure 4C). While we noted no significant two-way ANOVA interaction of DOI and CREBαδ KO genotype, we observed a significant main effect of DOI [F₍₁,₁₂₎ = 58.64, p < 0.0001]. This indicates that the loss of CREBαδ isoforms does not alter either the expression of the 5-HT_2A receptor, or the behavioral HTRs evoked by the 5-HT_2A receptor agonist DOI, that are critically dependent on the cortical 5-HT_2A receptor.

We next performed in situ hybridization and qPCR analysis to address whether the regulation of specific neuronal plasticity-associated genes that exhibit pCREB enrichment at their promoters following DOI treatment, were altered in the neocortex of CREBαδ KO mice. Radioactive in situ hybridization analysis indicated that the DOI-evoked robust induction of Arc mRNA levels both in the somatosensory cortex and in the prefrontal cortex was significantly attenuated in CREBαδ KO mice (Figure 4D). Two-way ANOVA analysis for Arc mRNA levels in the somatosensory cortex (Figure 4E) indicated a significant DOI by CREBαδ KO genotype interaction [F₍₁,₁₂₎ = 8.39, p = 0.013], as well as significant main effects of DOI [F₍₁,₁₂₎ = 16.81, p = 0.002] and CREBαδ KO genotype [F₍₁,₁₂) = 24.39, p = 0.0003]. The robust DOI-evoked upregulation of Arc mRNA in the somatosensory cortex of WT control mice was completely lost in the CREBαδ KO mice. Two-way ANOVA analysis for Arc mRNA levels in the prefrontal cortex (Figure 4F) indicated a significant DOI by CREBαδ KO genotype interaction [F₍₁,₁₁₎ = 15.73, p = 0.002], as well as significant main effects of DOI [F₍₁,₁₁₎ = 78.83, p = 0.0001] and CREBαδ KO genotype [F₍₁,₁₁₎ = 14.75, p = 0.003]. The robust DOI-evoked upregulation of Arc mRNA in the prefrontal cortex of WT control mice was significantly attenuated in the CREBαδ KO mice. No change was observed in the basal expression of Arc mRNA in either the somatosensory or prefrontal of CREBαδ KO mice, which did not differ from vehicle-treated WT controls.

qPCR analysis was carried out to assess whether the DOI-evoked upregulation of Bdnf1, Cebpb, cFos, and Egr2 mRNA expression was altered in the neocortex of CREBαδ KO mice following acute DOI administration (Figures 4G–J). Two-way ANOVA analysis for Bdnf1 mRNA levels in the neocortex indicated no significant DOI by CREBαδ KO genotype interaction (Figure 4G), however we did observe a significant main effect of DOI [F₍₁,₁₀₎ = 10.19, p = 0.01] and no main effect of CREBαδ KO genotype. Two-way ANOVA analysis for Cebpb mRNA levels in the neocortex (Figure 4H) indicated a significant DOI by CREBαδ KO genotype interaction [F₍₁,₁₂₎ = 6.413, p = 0.026], as well as a trend toward a main effect of DOI [F₍₁,₁₂₎ = 3.388, p = 0.09] and a significant main effect of CREBαδ KO genotype [F₍₁,₁₂₎ = 50.49, p = 0.0001]. Tukey’s post hoc group comparisons indicated that the CREBαδ KO mice exhibited a significant baseline decrease (p = 0.03) in Cebpb mRNA levels in the neocortex. Further, while DOI-evoked a robust and significant upregulation of Cebpb mRNA levels in WT animals (p = 0.04), this was completely lost in the CREBαδ KO mice. Cebpb mRNA levels in the DOI-treated WT cohort differed significantly from the DOI-treated CREBαδ KO mice (p < 0.0001). Two-way ANOVA analysis for cFos mRNA levels in the neocortex (Figure 4I) indicated a significant DOI by CREBαδ KO genotype interaction [F₍₁,₁₂₎ = 8.966, p = 0.011], as well as a trend toward a main effect of DOI [F₍₁,₁₂₎ = 4.493, p = 0.056] and a significant main effect of CREBαδ KO genotype [F₍₁,₁₂₎ = 102.5, p < 0.0001]. Tukey’s post hoc group comparisons indicated that the CREBαδ KO mice exhibited a significant baseline reduction (p = 0.001) in expression of cFos mRNA in the neocortex, and while DOI-evoked a robust and significant upregulation of cFos mRNA levels in WT animals (p = 0.02) this was completely lost in the CREBαδ KO mice. Neocortical c-Fos mRNA levels differed significantly between DOI-treated WT mice and the DOI-treated CREBαδ KO cohort (p < 0.0001). In contrast, we observed no significant DOI by CREBαδ KO genotype interaction for Egr2 mRNA levels within the neocortex (Figure 4J). Further, while we did note a significant main effect of DOI for Egr2 expression [F₍₁,₁₁₎ = 10.19, p = 0.003] we observed no main effect of CREBαδ KO genotype. Collectively, these observations indicate that CREB contributes to the acute DOI-evoked upregulation of Arc, Cebpb, and cFos mRNA within the neocortex, but not to the DOI-induced neocortical increase in Bdnf1 and Egr2 mRNA expression. Furthermore, baseline expression of Cebpb and cFos mRNA, but not Arc mRNA is also significantly attenuated in the CREBαδ KO mice. These results demonstrate that the transcription factor CREB contributes to the regulation of a subset of neuronal plasticity-associated genes that are regulated by the hallucinogenic 5-HT_2A receptor agonist, DOI in the neocortex.