Increasing Endocannabinoid Tone Alters Anxiety-Like and Stress Coping Behaviour in Female Rats Prenatally Exposed to Valproic Acid

Abstract

Given the sex differences evident in the prevalence of autism, there is an increased awareness of the importance of including females in autism research to determine sexual dimorphism and sex-specific treatments. Cannabinoids and endocannabinoid modulators have been proposed as potential novel treatments for autism-related symptoms; however, few studies to date have examined if these pharmacological agents elicit sex-specific effects. The aim of the present study was to use the valproic acid (VPA) model of autism to compare the behavioural responses of male and female rats and examine the effects of increasing endocannabinoid tone on the behavioural responses of VPA-exposed female rats. These data revealed that VPA-exposed male, but not female, rats exhibit reduced social responding in the three-chamber and olfactory habituation/dishabituation (OHD) test during adolescence. In comparison, VPA-exposed female, but not male, adolescent rats exhibited anxiety-like behaviour in the elevated plus maze (EPM) and open field test (OFT). In VPA-exposed female rats, increasing 2-AG levels augmented anxiety-like behaviour in the EPM and OFT, while increasing AEA levels reduced stress coping behaviour in the swim stress test. These data highlight sexual dimorphic behaviours in the VPA model and indicate that enhancing endocannabinoid levels may exacerbate negative affective behaviour in VPA-exposed females. Thus, considerations should be paid to the possible sex-specific effects of cannabinoids for the treatment of symptoms associated with autism.

Keywords: 2-AG; FAAH; MGL; VPA; anandamide; autism; sex differences; social behaviour.

Figure 1

The effect of prenatal VPA exposure on social behaviour in male and female rats. (a) Latency to reach home bedding in the nest-seeking test at PND 13 (n = 7 to 10 per group). (b) Time interacting with animal in the sociability phase of the three-chamber test during adolescence (n = 10 to 11 per group). (c) Time interacting with novel animal in the social novelty preference phase of the three-chamber test during adolescence (n = 12 per group). (d) Time spent sniffing each scent (water, lemon, same sex, opposite sex), (e) discrimination index ((same sex)/(same sex + lemon) × 100) and (f) total time spent sniffing the same sex scent in the OHD test during adolescence (n = 7 to 12 per group). (g) Total social interaction during the 10 min DSI test and the first min of the trial and (h) chasing, climbing and pinning behaviour during the DSI test during adolescence (n = 6 per group). (i) Unified behavioural social score (n = 12 per group). Data expressed as mean + SEM. * p < 0.05 vs. saline-exposed males; ⁺ p < 0.05 vs. saline-exposed females.

Figure 2

The effect of prenatal VPA exposure on anxiety-like behaviour and recognition memory in male and female adolescent rats. (a) Time in open arms and (b) number of entries into the open arms during the EPM (n = 12 per group). (c) Time in inner zone and (d) number of entries in the inner zone during the OFT (n = 12 per group). (e) Unified behavioural anxiety score (n = 12 per group). (f) Locomotor activity in open field arena (n = 12 per group) and (g) time interacting with familiar and novel objects during the NOR trial (n = 11 to 12 per group). Data expressed as mean + SEM. * p < 0.05 vs. saline-exposed males; ⁺ p < 0.05, vs. saline-exposed females. ^## p < 0.01 vs. corresponding group interacting with object 1 and 2.

Figure 3

The effect of PF3845 (10 mg/kg) and MJN110 (5 mg/kg) on nociceptive and affective behaviour and endocannabinoid levels in VPA-exposed female rats during adolescence. (a) Latency to respond in the HPT. (b) Time in open arms and (c) number of entries into the open arms during the EPM. (d) Locomotor activity, (e) time in inner zone and (f) number of entries into the inner zone during the OFT. (g) Time spent immobile during the 15-min FST and (h) over-5-min time bins. (i) AEA and (j) 2-AG levels in the cortex. Data expressed as mean + SEM (n = 11 to 12 per group). * p < 0.05 vs. vehicle.

Figure 4

Schematic depicting the effects of FAAH and MGL inhibition on anxiety-like behaviour in VPA-exposed female and male rats. Bold highlights the data depicted in the current study. References provided for data cited in published literature.

Figure 5

Schematic of the experimental design for the behavioural characterisation of male and female rats prenatally exposed to saline or VPA (experiment 1a–b) and examining the effect of enhancing AEA and 2-AG tone on nociceptive and affective behaviour in VPA-exposed female rats (experiment 2).