Fig. 1. Calcyon transgenic mice. (A) Map of TRE-FLAG-hCalcyon transgene construct. The calcyon transgene contains the tetracycline transactivator (tTA) binding site (TRE) and an RNA polymerase II transcriptional start site of the cytomegalovirus IE promoter upstream of the FLAG epitope fused to full-length human calcyon cDNA, followed by a β-globin polyadenylation sequence. (B) PCR screen of tail DNA to identify transgenic progeny. (C) Representative image from a 1.7 μm thick optical section through the CA1 region of the hippocampus of TRE-Cal/tTA (Cal^OE) mouse expressing human calcyon immunolabeled for calcyon (red) and NeuN (green). Nuclei (blue) were stained with a DNA-specific dye (Sytox Green). Note that calcyon is expressed in neurons (NeuN-positive cells). Scale bar is 50 μm. (D) Immunolabeling of hCalcyon is not detected in hippocampus of either the TRE-Cal or αCamKII-tTA (tTA) single transgenic mice.

Fig. 2. Calcyon transgene expression is observed in the prefrontal cortex striatum and amygdala of Cal^OE mice. Human calcyon (red) is clearly detected in the cytoplasm around neuronal nuclei (green) of the forebrain, including the medial prefrontal cortex (mPFC), the basolateral amygdala (BLA), and the dorsal striatum (CPU). Note that glia-like nuclei which appear small and uniformly stained (white arrowheads [27]), do not express the calcyon transgene. Each image represents 180 μm × 150 μm of tissue.

Fig. 3. Cal^OE mice display increased basal locomotor activity. (A) Bars show cumulative distance traveled (cm) per zone and in entire box (total) during the 30 min open field test session by the Cal^OE (dark bars) and tTA (light bars) mice. (B) Average ambulatory counts (horizontal photo beam breaks) measured per 5 min segment (bin). *p < 0.05; **p < 0.01 based on paired t-test comparison of tTA and Cal^OE values.

Fig. 4. Cal^OE mice exhibit increased exploratory activity and reduced anxiety. (A) Bars show mean time (s) spent in the light and dark areas during the 10 min light–dark box test by the Cal^OE (dark bars) and tTA (light bars) mice. *p < 0.05 and **p < 0.01, paired t-test.

Fig. 5. Cal^OE mice exhibit increased exploratory activity as well as reduced anxiety. (A) Bars show the mean and error bars the S.E.M. of time spent (s) in each area of the elevated plus maze by the tTA (light bars) and Cal^OE (dark bars) mice during a 10 min test session. (B) Exploratory activity data show the mean number of open and closed arm entries, of times the subjects head extended over the edge of the open arm (head dip) or times the subject traveled to the end of the open arm (end explore). (C) Mean distance traveled in each area of the maze individually and combined (total). *p < 0.05 and **p < 0.01, paired t-test.