Altered motor, anxiety-related and attentional task performance at baseline associate with multiple gene copies of the vesicular acetylcholine transporter and related protein overexpression in ChAT::Cre+ rats

Abstract

Transgenic rodents expressing Cre recombinase cell specifically are used for exploring mechanisms regulating behavior, including those mediated by cholinergic signaling. However, it was recently reported that transgenic mice overexpressing a bacterial artificial chromosome containing choline acetyltransferase (ChAT) gene, for synthesizing the neurotransmitter acetylcholine, present with multiple vesicular acetylcholine transporter (VAChT) gene copies, resulting in altered cholinergic tone and accompanying behavioral abnormalities. Since ChAT::Cre+ rats, used increasingly for understanding the biological basis of CNS disorders, utilize the mouse ChAT promotor to control Cre recombinase expression, we assessed for similar genotypical and phenotypical differences in such rats compared to wild-type siblings. The rats were assessed for mouse VAChT copy number, VAChT protein expression levels and for sustained attention, response control and anxiety. Rats were also subjected to a contextual fear conditioning paradigm using an unconditional fear-inducing stimulus (electrical foot shocks), with blood samples taken at baseline, the fear acquisition phase and retention testing, for measuring blood plasma markers of hypothalamic-pituitary-adrenal gland (HPA)-axis activity. ChAT::Cre+ rats expressed multiple mouse VAChT gene copies, resulting in significantly higher VAChT protein expression, revealed anxiolytic behavior, hyperlocomotion and deficits in tasks requiring sustained attention. The HPA-axis was intact, with unaltered circulatory levels of acute stress-induced corticosterone, leptin and glucose. Our findings, therefore, reveal that in ChAT::Cre+ rats, VAChT overexpression associates with significant alterations of certain cognitive, motor and affective functions. Although highly useful as an experimental tool, it is essential to consider the potential effects of altered cholinergic transmission on baseline behavior in ChAT::Cre rats.

Keywords: 5-CSRTT; ChAT::Cre rats; Cre-recombinase; HPA endocrine axis; Touchscreen-based behavioral testing; Vesicular acetylcholine transporter.

Fig. 1

The rats’ behavior was assessed for 10 min in the OF arena to measure locomotion and anxiety-like behavior. a The timeline of the anxiety-related and locomotion test procedures performed on the rats are shown; all rats performed the same behavioral tests in the order shown. To investigate the acute effect of foot shock at post-acquisition and post-retention stage on plasma corticosterone, leptin and glucose levels, blood samples were collected at the time-points shown, to compare to baseline levels. In the OF test, the behavioral profile of ChAT::Cre+ rats (n = 8) differed from their ChAT::Cre− siblings (n = 10). Taken together, the parameters b average speed of movement, c total distance traveled, d number of rearings made and also e the time spent rearing during the test, indicated that ChAT::Cre+ rats were hypermobile. Other parameters like f number of entries into inner field and (g) time spent in the inner field, further revealed that ChAT::Cre+ rats were in a mixed hypermobile and anxiolytic state. All data are presented as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 and ***p < 0.001, compared to ChAT::Cre− rats

Fig. 2

Anxiety-like behavior in the rats was further assessed in the EPM maze, where the task is based on an approach-avoidance conflict and greater avoidance of the open arms is interpreted as an exhibition of higher anxiety levels. The findings reflected those obtained for OF testing, with regards to both general ambulation and anxiety-like behavior. Over the duration of the test (5 min), ChAT::Cre+ rats (n = 8) traveled on average at a greater speed and b covered a greater distance than the ChAT::Cre− rats (n = 10). Compared to ChAT::Cre− rats, ChAT::Cre+ ones also revealed hypermobility by making c significantly more rearing bouts and d spending more time rearing. ChAT::Cre+ rats also made e more entries into the open arms of the EPM apparatus and f spent more time in the open arms than their ChAT::Cre− siblings, reflecting the general anxiolytic profile detected for the ChAT::Cre+ rats seen during testing in the OF. All data are presented as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 and ***p < 0.001, compared to ChAT::Cre− rats

Fig. 3

The LDB is another conventional anxiety test that is based on the innate aversion of rodents to brightly illuminated areas, which was utilized to test unconditional anxiety responses in the rats. The ChAT::Cre+ rats (n = 8) showed a greater preference for the light compartment of the box compared to their ChAT::Cre− siblings (n = 10). Behavioral parameters assessed were: a total number of entries made into the light chamber, b time spent in the light chamber and c latency time for rats to enter the light chamber. All data are presented as the mean ± SEM. *p ≤ 0.05, compared to ChAT::Cre− rats

Fig. 4

Performance in the 5-CSRTT assessed for differences in attention, impulsivity and cognitive flexibility between the transgenic and Wt rats. ChAT::Cre+ rats (n = 13) made significantly more omission errors and fewer perseverative responses than their ChAT::Cre− siblings (n = 8) during 5-CSRTT testing, which measured the following parameters: a choice accuracy; b average number of correct responses; c average number of incorrect responses; d latency to make a correct nose poke response following stimulus presentation; e latency to make an incorrect response following stimulus presentation; f average number of premature responses; g latency to collect sugar pellet reward from food magazine after a correct response; h average number of errors of omission and i average total number of perseverative responses. All data are presented as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, compared to ChAT::Cre− rats

Fig. 5

A version of the 5-CSRTT, where SD length was varied (0.6, 0.3 and 0.15 s), further measured attentiveness, impulsivity, processing speed and distractibility in the rats. SD length variance exerted an overall significant effect on a choice accuracy. This was since, compared to ChAT::Cre− rats (n = 8), the ChAT::Cre+ ones (n = 13) made on average, b less correct responses and, therefore, c more incorrect responses, as SD decreased. d ChAT::Cre+ rats made less perseverative responses compared to ChAT::Cre− ones, with the number of perseverative responses that decreased most significantly at the longest duration probe, namely 0.15 s. e As detected in the standard 5-CSRTT, a tendency by ChAT::Cre+ rats to omit more responses compared to baseline behavior was not repeated. f Groups revealed similar levels of premature responses. All data are presented as the mean ± SEM. **p ≤ 0.01, compared to ChAT::Cre− rats

Fig. 6

Freezing behavior and blood plasma analysis of HPA-axis biomarkers performed on ChAT::Cre+ (n = 5) and ChAT::Cre− (n = 8) rats at baseline (2 days before initiating the task protocol), with testing that was then repeated at the fear acquisition and fear retention stages of a CFCT. a Freezing scores acquired for retention testing; b comparison of corticosterone blood plasma levels between genotypes at the different CFCT testing phases; c a similar comparison made for glucose blood plasma levels and for d leptin blood plasma levels. The significant increase in blood plasma corticosterone levels seen in both rat genotypes from baseline to a repeat analysis soon after rats completed the fear acquisition and also the fear retention test phases, indicates that all rats of this strain, regardless of the genotype (ChAT::Cre+ vs. Wt), respond in a biologically intact manner to aversive conditioning. All data are presented as the mean ± SEM. *p ≤ 0.05 and ***p < 0.001, compared to both genotype rats’ baseline measurements

Fig. 7

Measurement of prefrontal cortical VAChT mRNA and protein levels. a qPCR results indicated additional mouse VAChT gene copies in all ChAT::Cre+ rats (n = 5) compared to ChAT::Cre− controls (n = 8). This translated to b increased mouse VAChT mRNA expression in Cre+ (n = 5) and Cre− (n = 7) siblings compared to Wt mice (n = 4) and also c increased VAChT protein expression in PFC brain samples of Cre+ (n = 5) and Cre− (n = 8) rats. All data are presented as the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 and ***p < 0.001, compared to ChAT::Cre− rats and Wt mouse