Neuronal functions of long noncoding RNAs (lncRNAs) are poorly understood. Here we describe identification and function of lncRNA GM12371 in regulating synaptic transmission, synapse density, and dendritic arborization in primary hippocampal neurons. GM12371 expression is regulated by cAMP signaling and is critical for the activity regulated synaptic transmission. Importantly, GM12371 is associated with transcriptionally active chromatin and regulates expression of several genes involved in neuronal growth and development. Taken together, these results suggest that GM12371 acts as a transcriptional regulator of synapse function.

Despite the growing evidence suggesting that long noncoding RNAs (lncRNAs) are critical regulators of several biological processes, their functions in the nervous system remain elusive. We have identified an lncRNA, GM12371, in hippocampal neurons that is enriched in the nucleus and necessary for synaptic communication, synapse density, synapse morphology, and dendritic tree complexity. Mechanistically, GM12371 regulates the expression of several genes involved in neuronal development and differentiation, as well as expression of specific lncRNAs and their cognate mRNA targets. Furthermore, we find that cAMP-PKA signaling up-regulates the expression of GM12371 and that its expression is essential for the activity-dependent changes in synaptic transmission in hippocampal neurons. Taken together, our data establish a key role for GM12371 in regulating synapse function.

One of the most important challenges in modern molecular neurobiology is to understand the dialogue between genes and synapses. Decades of studies have led to the identification of key molecular players that govern synapse function. For example, neurotransmitter receptors at postsynaptic compartments (1⇓–3), synaptic vesicle release machinery at presynaptic compartments (4, 5), various pre- and postsynaptic scaffolding proteins (1⇓–3, 6), transsynaptic signaling (7), and translation machinery at the synapse (8⇓–10) have been identified. In the nucleus, expression of specific genes and remodeling of chromatin (11⇓⇓⇓⇓–16) are known to regulate synapse function. However, the molecular underpinnings of how changes in gene expression results in regulating synapse function remain to be understood in detail. Particularly, we know little about the contribution of the noncoding transcriptome in regulating synapse function. The noncoding transcriptome is highly diverse and includes ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) (17⇓⇓⇓⇓–22). Among these, the neurobiology of lncRNAs remains the least understood. A large number of lncRNAs have now been discovered as a consequence of unbiased sequencing of genomes and transcriptomes (23⇓⇓⇓–27).

lncRNAs are transcripts that are more than 200 nucleotides long and are important regulators of gene expression (23⇓⇓⇓⇓–28). An increasing number of functional studies have established that lncRNAs regulate almost every stage of gene expression, from epigenetic modifications in the nucleus (29⇓–31) to messenger RNA (mRNA) stability (32⇓–34) and translation (35) in the cytoplasm. Recent studies have also profiled lncRNA metabolism profiles relative to mRNAs, demonstrating that lncRNAs differ in their half-lives, posttranscriptional modifications, subcellular localization patterns, and sequence conservation (19, 36). Notably, while the promoters for lncRNAs show high sequence conservation, the gene products show less conservation, although the presence of lncRNA orthologs across species (37, 38) have been documented. Despite this trend, lncRNAs have been shown to hold important regulatory functions in mammalian neurons. Previously, it has been shown that Malat1—a 7-kb single exon lincRNA—is highly enriched in nuclear speckles in the mouse hippocampus. This transcript, while showing low sequence conservation between rodents and humans, regulates alternative splicing of genes involved in nuclear organization and neuronal function (39). It has also been shown that a natural antisense transcript of BDNF can regulate BDNF expression in the mouse hippocampus, and shows only partial conservation within the BDNF overlapping region (40). Furthermore, the noncoding RNA BC1 regulates fragile X mental retardation protein-mediated protein synthesis in dendrites (41). While these discoveries suggest the significance of lncRNAs in the brain, fundamental gaps remain in our understanding of whether and how lncRNAs regulate synaptic transmission, synaptic architecture, and synaptically relevant protein-coding gene expression.

Here we describe the identification and characterization of an lncRNA, GM12371. We find that GM12371 is critical for synapse function in hippocampal neurons. Inhibition of its function resulted in a decrease in synaptic transmission, total synapse density, number of mushroom spines, and dendritic arborization. We also identified molecular targets of this lncRNA; among them are two lncRNAs that regulate expression of synaptically pertinent mRNAs in cis. Furthermore, we find that expression of GM12371 is regulated by cAMP-PKA signaling and that expression of GM12371 is necessary for the activity-dependent changes in synaptic transmission.

Results

Discovery of GM12371. Our previously published work has uncovered differentially expressed lncRNAs in the hippocampus, as well as hippocampal subregions (23). Using lncRNAs identified in this study, we employed a candidate approach by quantitative real-time PCR (qPCR) to identify lncRNAs expressed in mouse primary hippocampal neurons. We identified lncRNA GM12371, transcribed from chromosome 4 (Fig. 1A), as expressed in primary hippocampal neurons. We next confirmed its neuronal expression by FISH using a digoxigenin (DIG)-labeled 300-nt-long riboprobe (SI Appendix, Fig. S1A) and found that GM12371 is mostly localized in the nucleus. Fig. 1. Expression of lncRNA GM12371 is required for sEPSCs of synapses of hippocampal neurons. (A) Chromosomal position of GM12371. (B) Analysis of knockdown of GM12371 by three different gapmeRs and a gapmeR mix. A nontargeting gapmeR was used as specificity control. Expression level of actin mRNAs was used to assess nonspecific targeting effect of gapmeRs. Error bars are SEM, ***P < 0.0001, one-way ANOVA followed by Tukey test. (C) FISH analysis of knockdown of GM12371 by gapmeR B3 in primary hippocampal neurons. A nontargeting gapmeR was used as control for knockdown. A DIG-labeled riboprobe (SI Appendix, Fig. S1) was used to visualize cellular localization of GM12371. Two representative confocal projection images are shown for each condition. (Scale bars, 20 µm.) (D) Quantification of FISH data shown in C. Number of neurons analyzed: nontargeting gapmeR control = 166, GM12371 knockdown = 180, Error bars are SEM, ***P < 0.0001, Student’s t test. (E) Experimental outline for patch clamp electrophysiology to measure sEPSCs. (F) Two representative traces of sEPSC measurements in hippocampal neurons following control or GM12371 knockdown (KD) using gapmeRs. (G) Representative traces of sEPSCs recording show a decrease in sEPSC amplitudes in gapmeR B2 (green) and gapmeR B3 (red) groups relative to the control gapmeR (black). (H and I) Quantitation of changes in sEPSC amplitude and frequency, respectively. Bar graphs show percent changes in amplitude/frequency of sEPSCs. Error bars are SEM, *P < 0.05, ***P < 0.0001, one-way ANOVA followed by Tukey test. (J and K) Cumulative probability of changes in sEPSC amplitude and frequency respectively following GM12371 knockdown by gapmeRs. (Control gapmeR, n = 37; gapmeR B2, n = 35; gapmeR B3, n = 35). HP, hippocampus. To explore the potential role of GM12371, we first asked whether its expression is necessary for synaptic communication in primary hippocampal neurons. We carried out loss-of-function experiments by depletion of GM12371 using locked nucleic acid long RNA gapmeR oligonucleotide (gapmeR)-assisted knockdown (42, 43) in hippocampal neurons. To assess the efficiency of knockdown, we used three different gapmeRs against GM12371, as well as a mix that contained all three gapmeRs. A nontargeting gapmeR was used as a specificity control. qPCR analysis in Fig. 1B shows that the gapmeRs we synthesized could specifically knockdown GM12371 in hippocampal neurons [fold-change in GM12371 levels compared with control nontargeting gapmeR: 0.88 ± 0.012, gapmeR B1 0.32 ± 0.05, B2 0.19 ± 0.01, B3 0.12 ± 0.02, mix (B1 + B2 + B3) 0.27 ± 0.05; fold-change in actin mRNA levels compared with vehicle control: nontargeting gapmeR 1.5 ± 0.2, gapmeR B1 1.2 ± 0.2, B2 1.2 ± 0.0.14, B3 1.2 ± 0.0.13, mix (B1 + B2 + B3) 1.3 ± 013; n = 4 for all; P < 0.05, one-way ANOVA followed by Tukey test] (Dataset S1, Table S1). We then confirmed the gapmeR-mediated knockdown of GM12371 in hippocampal neurons using FISH analysis (Fig. 1 C and D) (percent mean fluorescence intensity of GM12371 staining in neurons following knockdown by gapmeR B3 compared with nontargeting gapmeR: 52.4 ± 8.7, P < 0.05; n = 20; unpaired two-tailed t test) (Dataset S1, Table S1). To examine whether GM12371 has a critical role in synaptic communication, we measured the effect of knockdown of GM12371 on spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings (Fig. 1 E and F). To knock down GM12371, we used two different gapmeRs (B2 and B3) (SI Appendix, Fig. S1B) identified from the above experiments. A nontargeting gapmeR was used as a specificity control. As shown in Fig. 1 F–K, we found that both gapmeRs produced a decrease in sEPSCs. Specifically, we observed a decrease in the amplitude and frequency of sEPSCs following GM12371 knockdown compared with the nontargeting gapmeR control (amplitude: control gapmeR, n = 37, 100 ± 7.8% vs. gapmeR B2 knockdown, n = 35, 72.12 ± 5.07%, and gapmeR B3 knockdown, n = 35, 66.24 ± 4.86%, P = 0.0003139, one-way ANOVA followed by Tukey test; frequency: control, n = 37, 100 ± 24.12%, gapmeR B2 knockdown, n = 35, 35.79.12 ± 8.52%, and gapmeR B3 knockdown, n = 35, 45.94 ± 11.29%, P = 0.01418, one-way ANOVA followed by Tukey test). Because the amplitude of EPSCs is related to the postsynaptic strength, whereas the frequency is correlated to the number of functional synapses and to the presynaptic release machinery, these results suggest that GM12371 has a significant role in excitatory synaptic transmission in hippocampal neurons. We then calculated the multiplicative shift in sEPSCs and found that sEPSC events from neurons with gapmeR B2 knockdown and with gapmeR B3 knockdown were decreased by a multiplicative factor of 0.72 and 0.66, respectively (compared with control gapmeR). It was previously shown that a multiplicative shift in the cumulative probability fraction of EPSC amplitudes is indicative of a cell-wide change in synaptic strength (44, 45). Taken together, these results suggest that normal expression of GM12371 is necessary for maintaining excitatory synaptic transmission in hippocampal neurons.

GM12371 Expression Regulates Spine Density and Dendritic Tree Complexity. The changes we observed in synaptic communication with GM12371 knockdown suggested specific disruptions at the synapse, such as reduction in spine density or a specific change in spine morphology. Alternatively, the reduction in synaptic transmission may be indicative of more subtle molecular defects at pre- and postsynaptic compartments. Therefore, to understand the mechanism underlying the regulation of synaptic transmission by GM12371, we first assessed whether knockdown of GM12371 can produce structural changes in hippocampal neurons. To address this, hippocampal neurons were transfected with pEGFPN1 to visualize neuronal architecture and confocal projection images were collected (Fig. 2A). Using confocal live cell imaging, we examined spine morphology and assessed spine density and spine type in GFP-labeled hippocampal neurons following knockdown of GM12371 by the two gapmeRs we used in the electrophysiology experiments. The image analyses (Fig. 2 B–D) showed that knockdown of GM12371 produced a decrease in total synapse density in hippocampal neurons, a specific decrease in mushroom and stubby spines, and an increase in thin spines (total spine density expressed as percentage of EGFP control; nontargeting gapmeR: 90.6 ± 1.6; gapmeR B2: 58.1 ± 1.7; gapmeR B3: 60.7 ± 3.1, P < 0.05, unpaired two-tailed t test; spine morphology: mushroom, stubby, thin spines: EGFP control 48.2 ± 5.5; 5.9 ± 1.8; 45.8 ± 3.9; nontargeting control gapmeR: 41.5 ± 2.8; 17.3 ± 3; 41.2 ± 3.8; gapmeR B2 25.5 3; 15.4 ± 4.6; 58.9 ± 2.1; gapmeR B3 30.6 ± 2.3; 16.5 ± 3.5; 52.9 ± 3.1 P < 0.05 for mushroom and thin spines, one-way ANOVA followed by Tukey test) (Dataset S1, Table S2) following GM12371 knockdown. Fig. 2. GM12371 regulates spine density, spine morphology, and dendritic tree complexity in hippocampal neurons. (A) Experimental outline. (B) Two representative confocal projection images of spines collected at different conditions are shown. (Scale bar, 20 µm.) Two different gapmeRs were used to knock down GM12371. A nontargeting gapmeR was used as control for knockdown. (C) Quantification of total spine density. Bar graphs show number of spines per 100 μm of distal dendrites quantified in EGFP control, control gapmeR, and gapmeR B2 and B3 knockdown neurons. (D) Quantitation of specific changes in spine morphology. Number of neurons analyzed for EGFP control = 20, gapmeR control = 18, gapmeR B2 = 17, gapmeR B3 = 24. (E) Sholl analysis to assess the effect of GM12371 knockdown on dendritic tree complexity. Shown are confocal projection images of EGFP expressing hippocampal neurons following transfection by nontargeting gapmeR (neg control), gapmeR B2 (GM12371 KD_B2), and B3 (GM12371 KD_B3) to specifically knock down GM12371. (Scale bars, 20 μm.) (F) Bar graphs show number of intersections at varying distances from soma. Number of neurons analyzed for gapmeR control = 18, gapmeR B2 = 18, gapmeR B3 = 25. Error bars are SEM, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Tukey test. Because we observed a major change in spine morphology with GM12371 knockdown, we asked whether GM12371 expression might be critical for dendritic tree complexity. As described in the spine analysis experiments above, hippocampal neurons were transfected with pEGFPN1 to visualize neuronal architecture and confocal projection images of dendritic arbors were collected for Sholl analysis (46). Intriguingly, GM12371 knockdown produced a significant reduction in dendritic tree complexity (Fig. 2 E and F) (distance from soma 30, 40, 50, 60, and 70 µm; number of intersections EGFP control: 35.2 ± 3,2; 47.6 ± 4.2; 47.8 ± 3.2; 46.4 ± 3; 37.2 ± 3.7; gapmeR B2: 18.1 ± 1.1; 25 ± 2.5; 27.7 ± 4; 26.4 ± 3.6; 17.1 ± 1.5; gapmeR B3: 23.2 ± 2.3; 27.2 ± 2.9; 26 ± 2.5; 31.4 ± 3.9; 23.75 ± 5.2, n = 12, P < 0.05, one-way ANOVA followed by Tukey test) (Dataset S1, Table S2). To assess whether the morphology changes that we observed in day in vitro (DIV) 17 neurons also occur in younger neurons that we used in the electrophysiology experiments, we carried out imaging analysis as described in Fig. 2. Data shown in SI Appendix, Fig. S2 suggest that expression of GM12371 is also required for dendritic arborization and spine density in DIV 10–12 neurons. Taken together, these results suggested that normal expression of GM12371 is essential for spine density and morphology and dendritic tree complexity.

GM12371 Is Associated with Transcriptionally Active Chromatin. Given its nuclear localization, we hypothesized that GM12371 regulates transcription by direct association with chromatin. We asked whether neuronal activation could lead to recruitment of GM12371 RNA to the transcriptionally active chromatin. Therefore, we carried out native chromatin immunoprecipitation (ChIP) (53) experiments and isolated both DNA and RNA from immunoprecipitated complexes (Fig. 4 A and B). Specifically, we analyzed the H3K27ac modification, which has been previously studied in activity-dependent hippocampal function (54). We immunoprecipitated H3K27ac complexes from hippocampal neurons exposed to forskolin that increased cAMP levels leading to transcriptional changes. Forskolin has been used to elicit chemically induced long-term potentiation in neuronal cultures (55) as well as in hippocampal slices (56). To assess the promoter of GM12371, we utilized publicly available ENCODE data from mouse early-postnatal forebrain H3K27ac ChIP (Gene Expression Omnibus accession no. GSE82428) and designed primers spanning the region upstream of the transcriptional start site with the most sequencing coverage. As a positive control, we also measured enrichment of the cFos promoter. IgG alone in ChIP assays were used to normalize data. We find that the GM12371 promoter was enriched in H3K27ac complexes immunoprecipitated from forskolin-treated neurons (Fig. 4C) (fold-change compared with DMSO, cFos: 1.9 ± 0.26; GM12371: 1.7 ± 0.14, n = 5; unpaired two-tailed t test, P < 0. 01). We then purified chromatin-associated RNAs from H3k27ac ChIP and assessed the abundance of GM12371 lncRNA using cFos RNA as a negative control. Consistent with its role in transcription, we find that GM12371 is enriched in transcriptionally active chromatin (Fig. 4D) (fold-enrichment in forskolin compared with DMSO 2.9 ± 0.39, n = 5, unpaired two-tailed t test, P < 0.01). Despite its being an immediate early gene, cFos RNA was not detected, indicating that the RNAs associated with the complexes are not nascent transcripts, but are likely regulatory. Fig. 4. Forskolin regulates the association of GM12371 to transcriptionally active chromatin. (A) Schematics of the native ChIP to assess transcriptionally active chromatin and associated RNAs. A histone marker of transcriptionally active chromatin, H3K27ac, was used in the ChIP analysis. (B) Experimental timeline for GM12371 knockdown and forskolin treatment for ChIP experiments. (C) qPCR analysis of GM12371 and cFOS promoter in ChIP. (D) qPCR analysis of GM12371 and cFOS RNA in ChIP. (E) qPCR analysis of GM12371 and GM10863 lncRNAs in the ChIP following the knockdown of GM12371 by gapmeR B3. A nontargeting gapmeR was used as a specificity control. All groups are normalized to IgG control, before indicated control group normalization. Bar graphs show fold-enrichment in the ChIP following forskolin stimulation. Error bars are SEM, n = 5, unpaired two-tailed t test, *P < 0.05, **P < 0.01. FSK, forskolin; Prm, promoter. We next asked whether knockdown of GM12371 by gapmeR would reduce the levels of GM12371 associated with transcriptionally active chromatin and whether its targets are also subsequently affected. As suggested earlier, the GM10863:Sox10 lncRNA:mRNA pair is a trans target of GM12371 (Fig. 3). Therefore, we assessed the association of GM10863 lncRNA (Fig. 3 C–E) to transcriptionally active chromatin after knockdown of GM12371. We found that knockdown of GM12371 resulted in a decrease in the association of both GM12371 and its target GM10863 lncRNA to transcriptionally active chromatin (Fig. 4E) (fold-change compared with knockdown using nontargeting control gapmeR, GM12371: 0.17 ± 0.07; GM10863: 0.13 ± 0.04; n = 5, unpaired two-tailed t test, P < 0.05). Taken together, these results suggest that GM12371 lncRNA associates with active chromatin to regulate transcription of its targets.

GM12371 Expression Is Necessary for the Activity-Dependent Changes in Synaptic Transmission. We next asked whether expression of GM12371 RNA is regulated by forskolin. qPCR analysis of the expression of GM12371 and two other lncRNAs randomly selected from our previous study (23) (GM11549, A43008) in SI Appendix, Fig. S7 shows that lncRNA GM12371 is up-regulated in response to exposure of 25 μM forskolin (fold-change 2.2 ± 0.3; n = 12; P < 0.05, one-way ANOVA followed by Tukey test) (Dataset S1, Table S14), whereas PKA inhibitor 14-22 amide blocked this up-regulation (fold-change 1.2 ± 0.03; n = 12; P < 0.05, one-way ANOVA followed by Tukey test) (Dataset S1, Table S14), suggesting cAMP-PKA signaling can regulate GM12371 levels in hippocampal neurons. Expression levels of two other lncRNAs were unchanged in primary hippocampal neurons with forskolin exposure (n = 12; P > 0.05) (Dataset S1, Table S14). We then investigated the temporal regulation of forskolin-induced GM12371 expression by carrying out a time course analysis (SI Appendix, Fig. S7B). We observed a significant change in GM12371 expression within 10 min of forskolin exposure; this then increased and peaked at 20 min, but persisted for 3 h after forskolin treatment [fold-change compared with DMSO (vehicle) 10 min, 1.9 ± 0.09, n = 3; 20 min, 3.6 ± 0.78, n = 3; 30 min, 3.11 ± 0.58, n = 6; 1 h, 1.96 ± 0.29, n = 4; 3 h, 1.86 ± 0.21, n = 3; 6 h, 1.23 ± 0.18, n = 3; P < 0.05 except for 6 h, unpaired two-tailed t test]. This activity-dependent expression pattern suggests that GM12371 acts as an immediate response gene in the cAMP-PKA signaling pathway. To further understand the up-regulation of GM12371 by cAMP signaling, we asked whether expression of GM12371 targets are also affected by activation of the cAMP signaling. Based on our data that gapmeR-mediated reduction in GM12371 levels resulted in corresponding changes in two lncRNA:mRNA pairs—GM10863:Sox10 and GM13292:PRKCq—we assumed that we would observe a change in the expression levels of GM10863:Sox10 and GM13292:PRKCq pairs in a GM12371-dependent manner in response to activation of cAMP-PKA signaling (SI Appendix, Fig. S7C). We assessed these possibilities by examining the expression of GM12371 and the two lncRNA:mRNA pairs following exposure to forskolin in the presence of GM12371 knockdown (Fig. 5 A and B). qPCR analysis shown in Fig. 5B suggest that consistent with the trans regulation of GM10863:Sox10 and GM13292:PRKCq pairs by GM12371, forskolin exposure produced an increase in the levels of lncRNAs as well as their cognate mRNAs (forskolin exposure alone mean fold-change ± SEM: GM12371, 1.9 ± 0.12; GM10863, 1.7 ± 0.15; SOX10, 1.9 ± 0.17; GM13293, 0.9 ± 0.14; PRKCq 1.7 ± 0.16; forskolin + nontargeting gapmeR control: GM12371, 2.2 ± 0.15; GM10863, 1.5 ± 0.0.09; SOX10, 1.5 ± 0.0.05; GM13293, 0.65 ± 0.16; PRKCq 1.6 ± 0.04; forskolin + GM12371 knockdown: GM12371, 0.49 ± 0.11; GM10863, 0.17 ± 0.19; SOX10, 0.51 ± 0.06; GM13293, 0.98 ± 0.09; PRKCq 0.4 ± 0.03, n = 6, unpaired two-tailed t test, P < 0.05 for comparison between forskolin alone/forskolin + control gapmeR vs. GM12371 gapmeR, expression changes were not significant for GM13293). Taken together, these results suggest that forskolin-induced changes in the GM10863:Sox10 pair depends on GM12371 levels, whereas regulation of the GM13292:PRKCq pair by forskolin might involve multiple mechanisms. However, in the basal condition, expression of the GM13292:PRKCq pair is dependent on GM12371 levels. Fig. 5. Expression of GM12371 is necessary for the forskolin-induced enhancements in synaptic transmission. (A) Experimental outline for assessing forskolin (FSK)-induced changes in GM12371 and its lncRNA:mRNA targets. (B) qPCR analysis of forskolin-induced changes in the expression of lncRNA:mRNA targets following GM12371 knockdown. Bar graphs show fold-changes. Data normalized to 18s rRNA levels. *P < 0.05, Student’s t test. (C) Experimental outline for assessing the necessity of GM12371 expression for forskolin-induced changes in synaptic transmission. (D) One representative sEPSC traces of control, forskolin, and two representative traces of forskolin + GM12371 knockdown (KD) are shown. (E) Representative sEPSC traces showing changes in amplitude in the presence of forskolin, forskolin + control KD, and forskolin + GM12371 KD. (F and G) Bar graphs showing percent changes in the amplitude and frequency following GM12371 KD in the presence of FSK. ****P < 0.0001, one-way ANOVA followed by Tukey test. (H and I) Cumulative probability analysis showing changes in the amplitudes and frequencies of sEPSCs. Because activation of cAMP-PKA signaling produced by forskolin exposure results in enhanced synaptic transmission in hippocampal neurons, we next asked whether expression of GM12371 is necessary for forskolin-induced changes in synaptic transmission. Following GM12371 knockdown, we measured sEPSCs before and after the treatment of forskolin (25 μM for 5 min) (Fig. 5 C–I). Quantitative analysis of sEPSC traces (Fig. 5 D and E) showed a decrease in sEPSC amplitudes (Fig. 5F) (mean ± SEM, nontargeting gapmeR control: 48.7 ± 10.93; GM12371 knockdown using gapmeR B3: 1.67± 3.02; P < 0.01, one-way ANOVA followed by Tukey test) and frequencies (Fig. 5G) (mean ± SEM: control knockdown: 172.12 ± 42.51; GM12371 knockdown: −1.71 ± 9.9; P < 0.01, one-way ANOVA followed by Tukey test). Consistent with these results, cumulative frequency analysis of electrophysiology data (Fig. 5 H and I) showed differences in both amplitude and frequencies, indicating that expression of GM12371 is necessary for the forskolin-induced changes in synaptic transmission in hippocampal neurons.