Focused Ultrasound Stimulates the Prefrontal Cortex and Prevents MK-801-Induced Psychiatric Symptoms of Schizophrenia in Rats

Abstract

Background and hypothesis: Treatment of schizophrenia remains a major challenge. Recent studies have focused on glutamatergic signaling hypoactivity through N-methyl-D-aspartate (NMDA) receptors. Low-intensity pulsed ultrasound (LIPUS) improves behavioral deficits and ameliorates neuropathology in dizocilpine (MK-801)-treated rats. The aim of this study was to investigate the efficacy of LIPUS against psychiatric symptoms and anxiety-like behaviors.

Study design: Rats assigned to 4 groups were pretreated with or without LIPUS for 5 days. The open field and prepulse inhibition tests were performed after saline or MK-801 (0.3 mg/kg) administration. Then, the neuroprotective effects of LIPUS on the MK-801-treated rats were evaluated using western blotting and immunohistochemical staining.

Study results: LIPUS stimulation of the prefrontal cortex (PFC) prevented deficits in locomotor activity and sensorimotor gating and improved anxiety-like behavior. MK-801 downregulated the expression of NR1, the NMDA receptor, in rat medial PFC (mPFC). NR1 expression was significantly higher in animals receiving LIPUS pretreatment compared to those receiving only MK-801. In contrast, a significant increase in c-Fos-positive cells in the mPFC and ventral tegmental area was observed in the MK-801-treated rats compared to those receiving only saline; this change was suppressed by pretreatment with LIPUS.

Conclusions: This study provides new evidence for the role of LIPUS stimulation in regulating the NMDA receptor and modulating c-Fos activity, which makes it a potentially valuable antipsychotic treatment for schizophrenia.

Keywords: LIPUS; NMDA receptor; c-Fos; mPFC; positive symptom.

Fig. 1.

Effects of LIPUS stimulation on the behavior of rats treated with MK-801. (A) Schematic diagram of the ultrasound transducer and treatment of the rat brain with LIPUS. The open field test was performed for 120 minutes for measuring (B) distance traveled, (C) progression in a 10-minute bin, and (D) time spent in the center area by the Sham group of rats and those treated with LIPUS or MK-801 or MK-801 plus LIPUS. (E) Effects of LIPUS on MK-801-induced disruption of prepulse inhibition. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,†, P < .05; **^,##,††, P < .01; ***^,###, P < .001; n = 10). .

Fig. 2.

LIPUS attenuates the MK-801-induced decrease in NR1 subunits and GAD65 in the mPFC of MK-801-treated rats. (A) Representative images of immunofluorescent labeling of NR1 subunits (red), PV (green), and DAPI (blue) in the mPFC of Sham, LIPUS-treated, MK-801-treated, and MK-801 + LIPUS-treated rats. The arrow indicates NR1- and PV-labeled cells. Images at high magnification show the methods used for quantification of NR1 puncta in PV interneurons. (B) Density of NR1 subunit puncta in PV interneurons in the mPFC. (C) Density of PV interneurons in the mPFC. Nuclei were counterstained with DAPI. Scale bars = 50 μm for all images. (D) Representative western blot bands and their quantification of GAD65 levels. (E) Representative western blot bands and their quantification of GAD67 levels. *, ^#, and ^† denote a significant difference from the Sham group, LIPUS group, and MK-801 group, respectively (**^,††, P < .01; ***^{,###,†††}, P < .001; n = 5). N.S., no significance; PV, parvalbumin; GAD, glutamic acid decarboxylase.

Fig. 3.

Effects of LIPUS stimulation on MK-801-induced c-Fos expression in the mPFC. (A) Western blot validation of c-Fos protein levels. (B) Representative images of c-Fos (red)- and NeuN (green)-positive cells in the mPFC. The arrow indicates c-Fos- and NeuN-labeled cells. Images at high magnification show the methods used for quantification of c-Fos in neurons. (C) A quantitative analysis shows that the LIPUS-increased expression of c-Fos is identifiable with neurons in the mPFC. (D) Density of NeuN+ cells in the mPFC. Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm for all images.*, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (**^,##,††, P < .01; n = 5). NeuN, Neuronal nuclei; N.S., no significance.

Fig. 4.

Effects of LIPUS stimulation on the VGLUT1/VGAT ratio and DAT level in MK-801-treated rats. Representative western blot bands and their quantification of (A) VGLUT1 levels and (B) VGAT levels. (C) The VGLUT1/VGAT ratio is derived from (A) and (B). (D) Representative western blot bands and their quantification of DAT levels. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,†, P < .05; ^††, P < .01; ***^,###, P < .001; n = 5). DAT, dopamine transporter; VGAT, vesicular gamma-aminobutyric acid transporter; VGLUT1, vesicular glutamate transporter 1.

Fig. 5.

Effects of LIPUS stimulation on MK-801-induced c-Fos expression in tyrosine hydroxylase (TH)-positive dopamine neurons in the ventral tegmental area (VTA). (A) Representative images of immunofluorescent labeling of c-Fos (red), TH (green), and DAPI (blue) staining in the Sham, LIPUS, MK-801, and MK-801 + LIPUS groups of rats in the VTA. The arrow indicates c-Fos- and TH-labeled cells. Images at high magnification show the methods used for quantifying c-Fos puncta in TH-positive dopamine neurons. (B) Quantitative analyses show that the MK-801-increased expression of c-Fos is identifiable with dopamine neurons in the VTA. (C) Density of TH-positive dopamine neurons in the VTA. Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm for all images. *, ^#, and ^† denote significant differences from the Sham group, LIPUS group, and MK-801 group, respectively (*^,#,†, P < .05; n = 5). N.S., no significance; TH, tyrosine hydroxylase.