Statistical analyses:
The principal behavioural measure was the mean percentage of correct
choices made across the blocks of 8 trials, for each experimental
condition. The behavioural data were analysed using multiple mixed-model
analyses of variance (ANOVAs), with the within-subject factor of drug
(saline vs clozapine) and between-subject factor of group (iDREADDs vs
GFP-control. Partial eta-squared
(ηp2 ) is reported as a measure
of effect size.
All data were screened for outliers, the assumptions of normality,
homogeneity of variances and covariances using SPSS Statistics 27(SPSS
Inc., Chicago, Ill., USA). A single outlier score (37.5%) was found for
just one animal for a single session (GFP group, Standard condition,
saline), and so this case remained in the analyses. Levene’s test based
on medians assessed the homogeneity of variance, showed that the
assumption was violated on the opposite-arm saline condition (p =
0.044) (Brown & Forsythe, 1974). No violations to the assumption of
homogeneity of covariance were found (all ps> 0.024) (see Tabachnick & Fidell, 2013). Where there was
a statistically significant interaction term, simple main effect
analyses were conducted using pooled error terms in JASP 14.1 (JASP
Team, 2022).
Multiple independent t-tests helped to compare control and baseline
scores, i.e., the pre-surgery training scores, post-surgery training
scores for each alternation condition prior to any infusions, as well as
for the infusion-free day scores between the clozapine infusions. These
analyses, applicable to Conditions 1-4, were to establish if the
performance of the iDREADDs and GFP-controls was statistically
comparable, prior to and between iDREADDs activations.
ResultsHistological findings:
Two criteria were required for inclusion in the experimental analyses.
First, the dorsal subiculum virus injections had to result in
appreciable bilateral label within granular retrosplenial cortex
(Figures 4, 5). Second, the infusion placements had to involve
retrosplenial cortex (Figure 3). Across both cohorts, a total of 6
iDREADDs and 8 GFP-control animals were excluded due to lack of viral
expression (unilateral or bilateral) in retrosplenial cortex (n=6),
off-target cannula placement (n=2) or both (n=6). Consequently, the
behavioural analyses derive from 14 iDREADDs and 8 GFP-control animals.
In four of these animals (n=3 iDREADDs; n=1 GFP) spread from the
anterior infusion cannulas may have reached the midcingulate cortex
(Vogt & Paxinos, 2012) as well as retrosplenial cortex. In some cases,
the virus injection spread into the dentate gyrus, which does not
directly innervate the retrosplenial cortex.