Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses

Abstract

In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.

Figure 1

Comparison of amino acid sequences of ApC/EBP from A. kurodai and A. californica. Amino acid sequences of cloned ApC/EBP are ∼97% homologous to those of A. californica. Unconserved amino acids are indicated by blackened squares. Basic region and leucine zipper domain are indicated by thick line. The long form of ApC/EBP has 52-amino acid insert marked by a shaded box.

Figure 2

Ectopic expression of ApC/EBP in cultured sensory neurons. Cultured sensory cells were injected with pNEXδ-ApC/EBP in the absence (middle) and presence (right) of double-strand RNA and were treated with five pulses of 5-HT 1 h after microinjection. (Left) A control cell without injection. The cultures were fixed and stained with anti-ApC/EBP antibody and Cy3-conjugated anti-mouse antibody 24 h after microinjection. Scale bar, 10 μm.

Figure 3

Transactivation by ApC/EBP of luciferase reporter gene driven by ERE. The expression of the luciferase reporter gene driven by ERE was increased by introduction of either the short form (ApC/EBP_S) or long form (ApC/EBP_L) of ApC/EBP constructs in Aplysia neurons. Normalized luciferase activity was obtained by dividing the luciferase activity by β-galactosidase activity. Results are expressed as mean ± SEM. Numbers in parentheses indicate the numbers of ganglia tested. (ERE-luci) ERE-luciferase; (CRE-luci) CRE-luciferase; (Δ-luci) enhancerless luciferase; (C/EBP_L) pNEXδ-ApC/EBP_L; (C/EBP_S) pNEXδ-ApC/EBP_S.

Figure 4

Effect of overexpression of ApC/EBP on long-term facilitation in the sensory-motor synapse. (A) Bar graph representing the effect of overexpression of ApC/EBP on long-term facilitation. Overexpression of ApC/EBP by injection of pNEXδ-ApC/EBP_L into the sensory cell produced long-term facilitation when it was combined with the application of 10 μM 5-HT for 5 min (one pulse, 1 ×) that can induce only short-term facilitation. The height of each bar corresponds to the mean percentage of change ± SEM in EPSP amplitude tested 24 h after 5-HT (10 μM) treatment. Numbers in parentheses represent the number of the sensory-to-motor synaptic connections tested. Plus (+) symbol in GFP and C/EBP rows indicates the successful expression of the microinjected DNAs (pNEXδ-GFPS65TΔC9 and pNEXδ-ApC/EBP_L) in the sensory cells as verified by GFP fluorescence, whereas minus (−) symbol indicates the failure of expression following the microinjection. In some experiments, the DNA construct(s) were not injected, indicated by the zero (0) symbol in the figure. 5-HT row indicates the number of 5-HT pulses treated in the cultures. A one-way analysis of variance and Duncan's multiple range test were used to determine the significance of the EPSP changes (F = 4.57, df = 5, P <0.002). (B) Examples of EPSPs recorded in motor neuron LFS after the stimulation of sensory neurons before (0 h) and 24 h after 5-HT treatment. The recording traces shown in the order of top to bottom in B are the representatives of the corresponding bars shown from left to right in A.

Figure 5

Inhibition of reporter gene expression by double-strand RNA. (A) Giant neurons in the abdominal ganglion were coinjected with pNEXδ-lacZ, pNEXδ-luciferase, and double-strand RNA of either luciferase or EGFP. Injection of luciferase double-strand RNA reduced luciferase activity in a sequence-specific and dose-dependent manner. pNEXδ-luciferase and double-strand RNA were mixed with the molar ratio of 1:0, 1:10, 1:100, and 1:1000 in the injection solutions. (•) Luciferase double-strand RNA; (○) EGFP double-strand RNA. (B) Sensory cells in the pleural ganglion were coinjected with CRE-lucifease and double-strand RNA luciferase and were treated by five pulses of 5-HT. CRE-driven luciferase gene expression, induced by five pulses of 5-HT, was completely blocked by luciferase double-strand RNA. Bars, mean ± SEM. Numbers in parentheses indicate the number of ganglia tested.

Figure 6

Inhibition by double-strand RNA of ApC/EBP expression blocks long-term facilitation in the sensory-motor synapse. (A) Bar graph representing the effect of ApC/EBP double-strand RNA on short-term (STF, open bars) and long-term facilitation (LTF, solid bars). Injection of either ApC/EBP double-strand RNA or antisense RNA blocked long-term facilitation induced by five pulse of 5-HT, but injection of luciferase double-strand RNA did not. In contrast, injection of ApC/EBP double-strand RNA, ApC/EBP antisense RNA, or luciferase double-strand RNA did not affect short-term facilitation induced by one pulse of 5-HT. The height of each bar corresponds to the mean percentage change ± SEM in EPSP amplitude tested after 5-HT treatment. (−) No injection of double-strand RNA. A one-way analysis of variance and Duncan's multiple range test were used to determine the significance of the EPSP changes by long-term facilitation (F = 21.99, df = 3, P <0.001). (B) Examples of EPSP recorded in motor neuron LFS after stimulation of the sensory neurons before (0 h) and 24 h after five pulses of 5-HT (10 μM) treatment. Control, no injection of double-strand RNA.