Acetylcholine elevation relieves cognitive rigidity and social deficiency in a mouse model of autism

Abstract

Autism spectrum disorders (ASD) are defined by behavioral deficits in social interaction and communication, repetitive stereotyped behaviors, and restricted interests/cognitive rigidity. Recent studies in humans and animal-models suggest that dysfunction of the cholinergic system may underlie autism-related behavioral symptoms. Here we tested the hypothesis that augmentation of acetylcholine (ACh) in the synaptic cleft by inhibiting acetylcholinesterase may ameliorate autistic phenotypes. We first administered the acetylcholinesterase inhibitor (AChEI) Donepezil systemically by intraperitoneal (i.p.) injections. Second, the drug was injected directly into the rodent homolog of the caudate nucleus, the dorsomedial striatum (DMS), of the inbred mouse strain BTBR T+tf/J (BTBR), a commonly-used model presenting all core autism-related phenotypes and expressing low brain ACh levels. We found that i.p. injection of AChEI to BTBR mice significantly relieved autism-relevant phenotypes, including decreasing cognitive rigidity, improving social preference, and enhancing social interaction, in a dose-dependent manner. Microinjection of the drug directly into the DMS, but not into the ventromedial striatum, led to significant amelioration of the cognitive-rigidity and social-deficiency phenotypes. Taken together, these findings provide evidence of the key role of the cholinergic system and the DMS in the etiology of ASD, and suggest that elevated cognitive flexibility may result in enhanced social attention. The potential therapeutic effect of AChEIs in ASD patients is discussed.

Figure 1

Intraperitoneal (i.p.) administration of Donepezil relieved cognitive rigidity in a dose-dependent manner. BTBR mice were injected i.p. with Donepezil (0.3 or 1.0 mg/kg) or saline (0.0 mg/kg) for 7 days before testing and 30 min before each testing day. (a) Scheme of the water T-maze test. On the first two days, mice had to learn and remember the location of the arm containing an escape platform (upper panel). On day 3, the platform was moved to the opposite arm (lower panel), and the reversal learning was tested in 2 consecutive days. (b) Percentage of correct turns leading to the platform taken in each day. (c) Scheme of the running/jammed wheel assay. Mice had 4 consecutive days of access to a running wheel (Run1–Run4, upper panel), which was later jammed for 2 consecutive days (Jam1–Jam2, lower panel). (d). Acquisition of the running habit was assessed by the latency to start running in each of the running days. (e) The ratio between interaction times in days Jam1 and Run4 (Jam1/Run4, left) indicates adjustment to the change. The ratio between interaction times in days Jam2 and Jam1 (Jam2/Jam1, right) indicates memory of the change. Data are presented as mean±SEM. *p<0.05, **p<0.01, N=8/dose.

Figure 2

Intraperitoneal (i.p.) administration of Donepezil improved sociability in a dose-dependent manner. BTBR mice were injected i.p. with Donepezil (0.3 or 1.0 mg/kg) or saline (0.0 mg/kg) for 7 days before testing and 30 min before each testing day. (a) Scheme of the three-chamber social preference test. One cage contained a stranger mouse (stranger cage) and the other was left empty (empty cage). (b) Sniffing durations of each wire cage. (c) Social preference index, calculated as (time with stranger)/(time with stranger+time with empty cage) × 100−50. (d) Scheme of the ‘social' running/jammed wheel assay. The preference of interaction with a stranger over an object was measured by comparing interaction time with a stranger mouse to interaction time with the previously-rewarding jammed-wheel (e). Social preference index (f) was calculated as (time with stranger)/(time with stranger+time with jammed wheel) × 100−50. (g) The male–male interaction test. Cumulative durations of sniffing and chasing the intruder mouse, as well as total social interactions, within a 15-min period. Data are presented as mean±SEM. ^#p=0.09, *p<0.05, **p<0.01, ***p<0.001, n.s: not significant. N=8/dose.

Figure 3

Intraperitoneal (i.p.) administration of Donepezil tended to improve social memory (preference for social novelty). BTBR mice were injected i.p. with Donepezil (0.3 or 1.0 mg/kg) or saline (0.0 mg/kg) for 7 days before testing and 30 min before each testing day. (a) Scheme of the three-chamber social novelty preference test. An unfamiliar stranger mouse (novel stranger) was put in the previously empty wire-cage. (b) Sniffing durations of the wire cage containing the novel stranger or familiar mouse. (c) Social novelty preference index, calculated as (time with novel stranger)/(time with novel stranger+time with familiar mouse) × 100−50. Data are presented as mean±SEM. ^#p=0.12, *p<0.05, **p<0.01, n.s: not significant. N=8/dose.

Figure 4

Diagrams illustrating the placement of the tips of the cannulae implanted to BTBR mice in experiment 2. For all mice, anterior–posterior placement was 1.00±0.05 mm anterior to bregma. Illustration modified from the coronal section 0.98 mm anterior to bregma in mouse brain atlas of Franklin and Paxinos (1997). aCSF, artificial cerebrospinal fluid; DMS, dorsomedial striatum; VMS, ventromedial striatum.

Figure 5

Direct Donepezil administration into the dorso-medial-striatum (DMS), but not into the ventromedial striatum (VMS), rescued cognitive rigidity and social deficiency. Mice were injected with artificial cerebrospinal fluid (aCSF) in the third running day for acclimation to procedure and reduction of stress. In each day from the fourth running day until the end of experiment, mice were injected with either aCSF or Donepezil (according to experimental group). (a) Latency to start running on the wheel in each of the running days, as a measure of habit acquisition. (b) Adjustment to change (ratio between interaction times with the wheel in days Jam1 and Run4, left) and memory of the change (ratio between interaction times with the wheel in days Jam2 and Jam1, right). (c) Social interaction: duration of interaction with an unfamiliar mouse (stranger) or with the jammed-wheel (wheel). (d) Social preference index, calculated as (time with stranger)/(time with stranger+time with wheel) × 100−50. DMS, dorsomedial striatum; VMS, ventromedial striatum; aCSF=0.5 μl artificial cerebrospinal fluid, Donepezil=100 ng Donepezil dissolved in 0.5 μl aCSF. Data are presented as mean±SEM. **p<0.01, n.s=not significant. aCSF to DMS: N=6. Donepezil to VMS: N=7. Donepezil to DMS: N=7.