Low-Frequency rTMS Ameliorates Autistic-Like Behaviors in Rats Induced by Neonatal Isolation Through Regulating the Synaptic GABA Transmission

Abstract

Patients with autism spectrum disorder (ASD) display abnormalities in neuronal development, synaptic function and neural circuits. The imbalance of excitatory and inhibitory (E/I) synaptic transmission has been proposed to cause the main behavioral characteristics of ASD. Repetitive transcranial magnetic stimulation (rTMS) can directly or indirectly induce excitability and synaptic plasticity changes in the brain noninvasively. However, whether rTMS can ameliorate autistic-like behaviors in animal model via regulating the balance of E/I synaptic transmission is unknown. By using our recent reported animal model with autistic-like behaviors induced by neonatal isolation (postnatal days 1-9), we found that low-frequency rTMS (LF-rTMS, 1 Hz) treatment for 2 weeks effectively alleviated the acquired autistic-like symptoms, as reflected by an increase in social interaction and decrease in self-grooming, anxiety- and depressive-like behaviors in young adult rats compared to those in untreated animals. Furthermore, the amelioration in autistic-like behavior was accompanied by a restoration of the balance between E/I activity, especially at the level of synaptic transmission and receptors in synaptosomes. These findings indicated that LF-rTMS may alleviate the symptoms of ASD-like behaviors caused by neonatal isolation through regulating the synaptic GABA transmission, suggesting that LF-rTMS may be a potential therapeutic technique to treat ASD.

Keywords: GABAAα1R; VGAT; autism spectrum disorders; low-frequency repetitive transcranial magnetic stimulation; mIPSCs; neonatal isolation.

Figure 1

Diagram of repetitive transcranial magnetic stimulation (rTMS) treatment and the modeled induced magnetic field of the circular coil. The Y064 is a parallel-wound solenoidal circular coil (inner radius = 9 mm, outer radius = 28.5 mm, height = 20.4 mm, wire cross-section = 18 mm², number of turns = 5 turns/layer × 6 layers = 30 turns). (A) The coil center was placed over the intersection of the midline and the interocular line of the rat (approximately 15 mm anterior to Bregma), which was hand-restrained in a suitable cloth sleeve. The windings of the coil covered an area between 6 mm anterior and 13.5 mm posterior to Bregma. (B) The heat map shows the modeled induced magnetic field with 100% of the maximum output of CCY-I, the actual measurement of which was 3.45 Tesla (0 mm below the surface of the circular coil center), 2.50 Tesla (5 mm below) and 1.58 Tesla (10 mm below), respectively.

Figure 2

Effects of low frequency (LF)-rTMS on social and repetitive behaviors caused by neonatal isolation. (A) Bar graph showing the ratio of time spent with the stranger animal vs. the object compartment in the three-chamber test. (B) Self-grooming within 10 min in an open-field arena. CTR: sham rTMS treated control rats without isolation; CTR + LF-rTMS: 14 day’s rTMS treated normal rats; ISO: sham rTMS treated rats with neonatal isolation; ISO + LF-rTMS: isolated rats treated with LF-rTM. Rats used in each group are n = 10–20. All data shown are represented as mean ± SEM (*P < 0.05, two-way analysis of variance (ANOVA) and Tukey’s post hoc test).

Figure 3

Effects of LF-rTMS on anxiety-/depressive-like behaviors caused by neonatal isolation. Time (A) and the number of entries (B) into the open and close arms in the elevated-plus maze test. Latency to immobility (C) and struggling time (D) in the forced swimming test. Rats used in each group are n = 11–24. All data shown are represented as mean ± SEM (*P < 0.05, **P < 0.01, two-way ANOVA and Tukey’s post hoc test).

Figure 4

LF-rTMS readjusts the balance between the excitatory and inhibitory synaptic activity of hippocampal pyramidal neurons in neonatally isolated rats. (A) Representative miniature inhibitory postsynaptic currents (mIPSCs) traces. Bar graphs of the mIPSC amplitude (B) and frequency (C). (D) Representative miniature excitatory postsynaptic currents (mEPSCs) traces. Bar graphs of the mEPSC amplitude (E) and frequency (F). Cells in each group are 14–31. Rats used are n = 5–8 per group. All data shown are represented as mean ± SEM (*P < 0.05, two-way ANOVA and Tukey’s post hoc test).

Figure 5

LF-rTMS down-regulates the increased expression of inhibitory synaptic receptors in the hippocampus caused by neonatal isolation. Sequential immunoblotting of synaptosomal fractions (A,B) and total tissue lysates (C,D) of hippocampal tissues collected from animals after behavioral tests. PSD-95 were used as the postsynaptic marker, and β-actin as the cytoplasmic marker. The relative protein level is normalized by the level of CTR. Rats used are n = 7–9 per group. All data shown are represented as mean ± SEM (*P < 0.05, **P < 0.01, two-way ANOVA and Tukey’s post hoc test).