Memory consolidation and gene expression in Periplaneta americana

Abstract

A unique behavioral paradigm has been developed for Periplaneta americana that assesses the timing and success of memory consolidation leading to long-term memory of visual-olfactory associations. The brains of trained and control animals, removed at the critical consolidation period, were screened by two-directional suppression subtractive hybridization. Screens identified neurobiologically relevant as well as novel genes that are differentially expressed at the consolidation phase of memory. The differential expression of six transcripts was confirmed with real-time RT-PCR experiments. There are mitochondrial DNA encoded transcripts among the up-regulated ones (COX, ATPase6). One of the confirmed down-regulated transcripts is RNA polymerase II largest subunit. The mitochondrial genes are of particular interest because mitochondria represent autonomous DNA at synapses. These transcripts will be used as one of several tools in the identification of neuronal circuits, such as in the mushroom bodies, that are implicated in memory consolidation.

Figure 1.

Visual associative learning. Experimental setup and training protocol. Restrained cockroaches were positioned at the center of the arena. The distance from the head to the position of visual and olfactory cues was 15 cm. A green LED as a visual cue was positioned in parallel with an odor cue ∼5° from the midline of the head. Under restricted sensory conditions, one eye and the ipsilateral antenna were covered (naive half), restricting sensory information to the opposite side (trained half). Training protocol comprised three pretraining trials, five training trials, and three testing trials.

Figure 2.

(A) Antennal projection responses (APRs) of cockroaches trained in nonrestricted sensory conditions (black bars) were tested for up to 24 h and compared with control/untrained animals (hatched bars). A high percentage of APRs were retained in animals trained in nonrestricted sensory conditions from 5 min to 24 h with no significant difference between intervals. Trained animals were significantly different from control animals. (B) APRs of cockroaches trained in restricted sensory conditions were tested for up to 24 h. APRs of the trained half were elicited at 5 min and retained up to 20 h, but show a marked decrease to pretraining levels at 24 h.

Figure 3.

Schema of forward subtraction to generate Trained-specific cDNAs. Patterns in the flowchart follow the progress of the subtraction. Opposing perpendicular patterns represent single-stranded cDNAs (ss-cDNA) that are specific to Trained#1 and T#2, respectively. Diamond pattern represents hybrids of T#1 and T#2 ss-cDNAs, i.e., cDNAs that are common in T#1 and T#2. These are Trained-specific cDNAs. For detailed description, see Results and Materials and Methods sections.

Figure 4.

Differential screening of T/C library clones. For screening with the dot blot technique, 48 clones are arrayed here in duplicates on nylon membranes to make two identical arrays. The membranes are hybridized with DIG-labeled T/C (A) or DIG-labeled C/T (B) cDNA pools. Arrowheads indicate cDNA clones that correspond to genes with increased expression in the brain of the trained roach.

Figure 5.

Differential screening of C/T library clones. For screening with the dot blot technique, 36 clones are arrayed here in duplicates on nylon membranes to make two identical arrays. The membranes are hybridized with DIG-labeled C/T (A) or DIG-labeled T/C (B) cDNA pools. Arrowheads indicate cDNA clones that correspond to genes with increased expression in the brain of the trained roach.

Figure 6.

Comparison of A5/neuropep with related peptides. The conceptual translation product of A5/neuropep is aligned with D. melanogaster adipokinetic hormone precursor (P17975) (Noyes and Schaffer 1990) and with B. discoidalis prepro-hypertrehalosemic hormone (U35277) (Lewis et al. 1997). Multiple sequence alignment was performed using PileUp (GCG, Wisconsin). The identical residues in the alignment were put on black background, and conserved subtitutions were put on gray (BoxShade server). Horizontal lines indicate the mature adipokinetic hormone (AKH) and hypertrehalosemic hormone (HTH).

Figure 7.

Relative transcript levels are expressed as Trained versus Control ratios (gray bars). T/C and C/T clones were quantified with real-time RT-PCR. Mean ± SEM representation. Ratio equals 1 indicates no detectable change. ^*P < 0.05, two-tailed t-test.

Figure 8.

PCR amplification of T/C and C/T transcripts (Tables 1, 2) with primer pairs described in Table 3 is analyzed on ethidium bromidestained agarose gel. Numbers at the left indicate size expressed as bp. All reactions resulted in one band of the expected size.