2. Experiment 1 Introduction

5.1 Open Field Findings

Mephedrone produced an overall increase in corner occupancy in the open field; as

well as a decrease in rearing, grooming, and center occupancy. Taken together, these results

are indicative of heightened anxiety, support Hypothesis 1, and are consistent with previous

studies examining anxiogenic drugs (e.g., Aitchison & Hughes, 2006; Pometlova et al., 2012;

Quinteros-Munoz et al., 2010; Thompson, 2012). While there was no statistically significant

change in freezing behaviours, the data does appear to be trending upwards with high doses

of mephedrone, which would similarly suggest increased anxiety.

The ambulation and walking measures also displayed significant increases following

treatment with mephedrone. While higher scores on these two measures do not typically

indicate increased anxiety, these results are consistent with the increase in locomotor activity

generally observed following treatment with comparable psychomotor stimulant drugs (e.g.,

Gatch et al., 2015; Sahakian, Robbins, Morgan, & Iverson, 1975). This increased locomotor

activity may also explain the lack of significant findings for the freezing behaviours measure.

The results for defecation, a measure thought to represent levels of emotionality

(Anderson & Hughes, 2008), show a significant decrease following treatment with

mephedrone, which runs contrary to the hypothesis and the other results. This unexpected

finding however, can potentially be explained by the methodology of this particular

experiment: because subjects were tested 20 minutes following drug administration (in order

to allow for the drug to take full effect) it is entirely plausible that these subjects, made

anxious by the oncoming drug effects, defecated in their temporary holding cages prior to

being placed in the testing apparatus. Conversely, the control subjects, experiencing no

anxiogenic effects of drug treatment and comfortable in their dark holding cages, would

instead defecate only after being introduced to the anxiety-inducing testing apparatus. Given

that the number of faecal boluses was counted only in the testing apparatus and not the

holding cages, it may be that this measure is not valid with the present methodology.

When looking at the results across the dose range tested; ambulation, walking, and

corner occupancy measures all show initial increases, peaking at 20mg/kg, before decreasing.

The corner occupancy measure appears to display the opposite (a decrease, followed by an

increase) though as lower levels of this measure represents more anxiety we can interpret this

result to be the same as the others. Taken together these results would appear to support

Hypothesis 2 (an inverted ‘U’ shaped dose-response curve), however, when the results for

males and females are examined separately an interesting phenomenon can be observed. The

predicted ‘U’ shaped curves for ambulation, center occupancy, and corner occupancy (later

found to be significant quadratic trends) are found for male subjects only. Female subjects,

conversely, appear to display a more linear dose-response relationship with higher doses of

mephedrone producing greater levels of anxiety-like behaviours. That this finding is

replicated (albeit inversely) in both the center and corner occupancy measures adds validity

to the detected effect, and suggests it is not simply an artefact of the data. The effect has not

been found in previous studies, which suggests that either it is unique to mephedrone, or

more likely, it is a consequence of the research literature’s traditional use of male-only

experimental designs (Hughes, 2007; Kokras & Dalla, 2014).

The inverted ‘U’ shaped curve found in previous research has been suggested to be a

result of stereotypy behaviours contaminating the behavioural measures at higher doses

(Clemens et al., 2006). This explanation is consistent with observations made by the

experimenter in the current study, wherein subjects on higher doses of the drug were

observed: tracing the outside walls of the apparatus (‘thigmotaxis’), turning repeatedly in

tight circles (‘gyration’), and engaging in repetitive head swaying behaviours. If performed

frequently enough, these behaviours could plausibly confound several of the behavioural

measures recorded in the open field: for example, one 60mg/kg subject spent the entire 5

minutes of the test running clockwise around the exterior boundary of the apparatus. This

animal would then be disproportionately likely to be recorded as having occupied a corner

square on a given observation, even though doing so was largely an artefact of increased

thigmotaxis rather than ‘hiding’ in the relative shelter of the corner. Center occupancy would

be likewise affected, as an animal tracing the boundary of the apparatus would have no

opportunity to be observed occupying the center squares.

These notes on stereotypy must be considered when interpreting the results of the

experiment, though they do not necessarily mean that the initial findings are invalid.

Increased anxiety may be strongly correlated with increased stereotypy, which would go

some way to explaining the inverted ‘U’ shaped curve: anxiety increases beyond the peak,

and accordingly so does stereotypy, potentially resulting in behaviours that erroneously

represent decreased anxiety. Furthermore, if females displayed fewer stereotypic behaviours,

or at least overtly responded in a different way, this theory could explain the unexpected sex

differences found in the dose-response relationships. It may be that higher doses of

mephedrone produce greater anxiety for both male and female subjects, though after a certain

point (in this case, 20mg/kg) the two sexes differ in their overtly observable behaviours

(namely, stereotypy).

In document The behavioural pharmacology of mephedrone (“bath salts”) and its effects on anxiety related behaviours in male and female hooded rats (Page 46-49)